Molecular Cell Biology Lesson 1 PDF

Molecular Cell Biology – Bolino Lesson 01 10th October 2024 Introduction The cell The cell is defined as the fundamental unit of organisms which can grow and proliferate (the name de...

Molecular Cell Biology – Bolino Lesson 01 10th October 2024 Introduction The cell The cell is defined as the fundamental unit of organisms which can grow and proliferate (the name derives from Robert Hooke 1665). The cell theory was first proposed in 1838-39 by Schleiden e Schwann and stated that all organisms originate from pre-existing cells from which they derive all the characteristics. In 1855 Rudolf Virchow proposed that omnis cellula e cellule, which indicates that every cell originates from another cell and thus according to the cellular theory of Schleiden-Schwann-Virchow: All living organisms are made of cells- Cells are the smaller structures of living organisms. New cells derive from pre-existing cells through cell division. Even if different cells have common features, all cells are characterized by: o Same types of macromolecules: proteins, DNA, RNA, sugars and lipids. o Similar structural aspects: plasma membrane, cytoplasm and nucleus. The limit of cell dimension is dictated by the ratio between surface and volume (surface/volume). If the volume is increasing, the surface cannot increase proportionally. Since the cells exchange solutes, gas and ions through the surface, a huge increase will affect the speed of exchanges, indeed, it would slow down the exchanges. Also, tissue-organization of cells is a way to increase surface, cell-cell junctions. The cell is made up 70% of water, that indeed is the fundamental composition of the cells. There are only 6 elements in the cell: H, C, O, N, S, P that build the monomers (small organic building blocks). Sugars are the monomers of polysaccharides, fatty acids of fats, amino acids of protein and nucleotides of nucleic acids. Molecules are built through covalent bonds, which are formed through condensation reaction (H2O removed); the covalent bond is broken with hydrolysis. Activated vectors Chemical-bond energy is stored in activated carrier molecules and is easy exchangeable; these molecules diffuse rapidly into cells. Activated vectors are something that carries energy and releases it through the break of a covalent bond (NAD, NADP, ATP, UDP, Acetyl-CoA etc.). Those reactions imply the building of macromolecules like DNA, RNA, sugars… We need energy and we take it from phosphate, CoA or oxidation (NAD/NADP). Trafficking This course is based on membrane trafficking and, in general, cellular trafficking between organelles: we talk about vesicular trafficking between organelles, but also about contact between organelles. Anyway, vesicles and organelles are different: In vesicles does not occur a reaction, they are only carriers surrounded by a membrane. Organelles are compartments in which occur a reaction; to regulate better this specific reaction, the organelle must be compartmentalized (such as like lysosome). Trafficking processes in cells are governed by the interaction between vesicles and the contact points between membranes and organelles. Introducing this concept reveals that vesicular trafficking is not a random process; rather, it relies on the cytoskeleton for directed movement. Vesicles must be transported to precise cellular locations, a process that is highly regulated. Although biological events occur on the millisecond scale, they maintain strict regulation. Common depictions of organelles in textbooks and scientific literature may not reflect actual cellular dynamics, as these representations are based on cells fixed onto slides and observed under microscopes. Such methods do not capture the true nature of cellular 1 Molecular Cell Biology – Bolino Lesson 01 10th October 2024 processes, which require live-cell imaging to accurately observe temporal dynamics. Observing trafficking in real time thus transforms our understanding, enabling the study of molecular interactions and events occurring within milliseconds. Plasma membrane Cellular processes involve the exchange of vesicles and the movement of materials within the cell, driven by precise molecular recognition. This recognition is enabled by specific chemical interactions. Cellular membranes, primarily composed of phospholipids, various lipids, and cholesterol, are assembled within the smooth endoplasmic reticulum. These membranes, particularly the plasma membrane (PM), are essential for maintaining cell shape and enabling the compartmentalization of vesicles and organelles. Although the chemical composition of the PM and other organelle membranes is similar, slight differences are crucial for facilitating distinct cellular functions. The plasma membrane also enables cell adhesion and intercellular communication, often mediated by integrins. Additionally, it serves as a selective barrier that separates the intracellular environment from the extracellular matrix, being generally impermeable except to small hydrophobic molecules and gases; transporters are therefore required for other molecules. Membrane properties, such as rigidity and fluidity, are influenced by cholesterol and the presence of double bonds in fatty acid chains, with higher cholesterol content contributing to increased rigidity and double bonds enhancing fluidity. Membrane asymmetry is essential, as it enables distinct chemical compositions across membrane layers, which is critical for cellular communication. Different classes of phospholipids—such as phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine—are distributed unevenly between the cytosolic and extracellular leaflets. Cholesterol distribution may also vary between these layers, and phosphoinositides are predominantly located on the cytosolic side. The extracellular surface of the plasma membrane (PM) is rich in sugar molecules bound to proteins and lipids, forming the glycocalyx; however, within organelles, sugars are situated on the cytosolic leaflet. Precise regulation across time and space is fundamental in biology. Interactions may occasionally occur through stochastic processes, where the local concentration of a molecule increases the likelihood of interaction with adjacent molecules. This “adjacency” is often created by mechanisms that actively position molecules in proximity. Another important concept in biology is polarization, where asymmetry is maintained across different sides of a structure. For instance, during vesicle budding, the inner leaflet of the membrane remains enclosed within the vesicle. However, upon fusion with a target membrane, this inner leaflet becomes exposed to the extracellular or external environment. This polarity is crucial for maintaining functional orientation and directional processes within cells. Membranes contain various embedded proteins, categorized based on their association with the lipid bilayer: Integral membrane proteins: These proteins are closely associated with the membrane and may be transmembrane but are not necessarily so. Peripheral membrane proteins: These proteins are not directly integrated into the membrane; instead, they attach to integral membrane proteins. 2 Molecular Cell Biology – Bolino Lesson 01 10th October 2024 Numerous proteins associate with membranes through lipid attachments, which serve as specific tags: Glycosylphosphatidylinositol (GPI): This lipid tag covalently binds to proteins intended for exposure to the extracellular space. Other lipid modifications, such as myristoylation, palmitoylation, and farnesylation, also serve as tags, anchoring proteins to membranes via covalent bonds formed with amino acid side chains. These proteins are typically inserted into membranes facing the cytosolic compartment (e.g., the inner leaflet of the plasma membrane or the external leaflet of vesicles). This tagging mechanism is key to recruiting specific proteins, such as Rab and other small GTPases, to membranes—a notable example of how activated GTPases are directed to their target locations within the cell. In addition to membrane asymmetry, membrane domains also play a critical role in directing cellular functions. A domain is a localized area within the membrane, enriched with specific proteins and lipids, which may differ on the internal or external face of the membrane. These domains enable the clustering of chemical entities that act as effectors, guiding and modulating the membrane’s biological roles. This organization allows the membrane to execute distinct functions in a spatially organized manner, enhancing its functional specificity and responsiveness. The cytoskeleton The cytoskeleton is essential for intracellular trafficking, and its structural components vary depending on cellular location and function. It comprises three primary classes of elements: Intermediate filaments (10 nm in diameter): Composed of monomers that form homophilic interactions, these filaments feature globular heads that can undergo post-translational modifications, such as phosphorylation on amino acid side chains (e.g., serine, threonine). This modification induces conformational changes in the globular heads, leading to filament expansion and reduction of space within the filament network. In neurons, for instance, these modifications can alter axon diameter, impacting the speed of action potentials. Intermediate filaments may be nuclear (like lamin, which influences nuclear shape and chromatin organization) or cytosolic, with some variations specific to cell types. Microtubules (25 nm in diameter): Formed from protofilaments of alternating α- and β-tubulin subunits, microtubules are polarized and hollow structures. The α-tubulin end, which grows more slowly, typically orients 3 Molecular Cell Biology – Bolino Lesson 01 10th October 2024 toward the cell center, while the faster-growing β- tubulin end points toward the periphery. Thirteen alternating α/β subunits make up each protofilament. Microtubules are highly dynamic, continuously polymerizing and depolymerizing unless stabilized by a GTP cap. They originate from the centrosome, the primary microtubule organizing center (MTOC). Notably, multiple microtubules can extend from a single centrosome. Actin microfilaments (7 nm in diameter): Essential for processes at the plasma membrane edge, actin filaments are formed by the polymerization of globular G-actin into filamentous F-actin. Actin- binding proteins, such as Arp2/3 and formin, facilitate polymerization in specific orientations. Arp2/3, for example, enables actin to polymerize at defined angles relative to existing filaments. The rearrangement of cytoskeletal filaments is crucial for cell movement, particularly across substrates like the extracellular matrix (ECM) or through endothelial barriers, as seen in cancer cell migration. These cytoskeletal filaments are crucial for enabling cellular motility, as cells extend several types of protrusions: Lamellipodia: These are 3D membrane protrusions where actin forms branched networks, facilitating broad, sheet-like extensions. Filopodia: These structures contain actin organized in tight, parallel bundles, supporting thin, finger-like projections. Stress fibers: These bundles of actin filaments are organized with myosin, forming networks that generate contractile forces within the cell. The formation of these structures is regulated by small Rho GTPases, including key players such as Rac1 and Cdc42. Cdc42 activation occurs when it binds GTP, a process mediated by GEF proteins. Activated Cdc42 can then recruit actin- binding proteins like formin, promoting actin polymerization that drives cell movement. The presence of Cdc42-GTP is both necessary and sufficient for initiating this process. Conversely, Rac1 activation leads to actin branching via the recruitment of the Arp2/3 complex, enabling lamellipodia formation. 4 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 Cell compartments and protein sorting Cell compartimentalization The central concept of cellular compartmentalization lies in the organization of materials to ensure their precise localization and timely availability, a process facilitated by various transport and localization mechanisms. Cellular compartments have several purposes, such as: 1. Restriction of Biochemical Reactions: Cellular compartments serve to localize and concentrate enzymes, substrates, and regulatory molecules, thus enabling specific biochemical reactions to occur in a controlled environment. 2. Expansion of Membrane Surface Area: Many reactions benefit from increased membrane area, as observed in processes like oxidative phosphorylation. For instance, mitochondria adapt by modifying their shape and expanding membrane surface area to meet the energy demands of the cell. Regulatory Complexity Introduced by Compartmentalization Although compartmentalization enhances regulatory control, it also complicates cellular processes due to the general impermeability of membranes. Since membranes typically do not permit the passage of most substances, specialized transport mechanisms are necessary to enable the movement of proteins and other critical molecules across compartment boundaries. These transport systems are crucial, as membranes inherently restrict the free movement of materials. This complexity, while adding organizational structure and precise regulation, introduces the need for specific transport and regulatory systems. Consequently, compartmentalization aids in maintaining distinct internal environments but requires advanced mechanisms to manage these boundaries effectively. Different types of intracellular compartments have been identified: 1. Nucleus and Cytosol: Connected by nuclear pore complexes that allow selective molecular exchange while maintaining nuclear-cytosolic separation. 2. Organelles in the Secretory and Endocytic Pathways: These include the endoplasmic reticulum, Golgi apparatus, endosomes, lysosomes, peroxisomes, and various connecting vesicles, which together facilitate protein modification, transport, and degradation. 3. Mitochondria: Specialized organelles with distinct internal structures supporting energy production and metabolic functions through compartmentalized reactions. Through this system, cells achieve efficient and organized functionality to support complex biochemical processes. Figure 1 Figure 2 5 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 The tables presented indicate the extent to which compartmentalization occupies space relative to the cell's total volume (Fig. 1) or as a percentage of the plasma membrane (Fig. 2). The relationship between cell volume and plasma membrane surface area is not directly proportional. An increase in cell volume does not necessarily result in a corresponding increase in the plasma membrane surface area. Certain cell types, such as pancreatic cells and hepatocytes, have adapted by developing a significantly larger proportion of membrane within their endoplasmic reticulum (ER)—both smooth and rough—relative to their plasma membrane. This adaptation facilitates their specialized functions. In contrast, other cell types contain substantially less membrane within their ER. As previously noted, observations in certain images, including those depicting mitochondria, may sometimes be misleading due to artifacts rather than true structural elements. Figure 3: An example of electron microscopy shows that certain organelles and structures appear darker than others; regions with higher density typically appear darker in the image. The darker appearance observed in electron microscopy images is a result of using fixed cells, as electron microscopy is typically performed on fixed samples. Although electron microscopy generally relies on fixed samples, the specific preparation and fixation techniques used can vary considerably, enabling improved image resolution. Certain organelles, such as lysosomes, appear darker in electron micrographs. This occurs because denser cellular structures absorb or scatter more electrons, resulting in a darker image. As the electron beam passes through the sample, electron-dense areas absorb or deflect a greater number of electrons. Lysosomes, for instance, are densely packed with a variety of enzymes—approximately seventy distinct types, though not limited to this number—that facilitate the breakdown and metabolism of different substances. Within each lysosome, multiple copies of these enzymes contribute to their high density, explaining why lysosomes appear darker than other cellular structures in electron microscopy images. By contrast, the cytoplasm appears lighter, as it is primarily composed of water, ions, and other less dense molecules. In cell biology, a fundamental concept is that most cellular compartments, or organelles, are membrane-bound. Common examples include mitochondria, the nucleus, and the lysosomal system, all of which are separated from the cytoplasm by membranes. However, recent advances have identified biomolecular condensates, which are distinct cellular compartments that lack surrounding membranes but still perform specialized functions. This emerging concept demonstrates that not all cellular compartments require a membrane to remain functionally distinct from the rest of the cell. Why are they defined as compartments? Biomolecular condensates are defined as compartments because they facilitate chemical reactions within a concentrated environment. The term "biomolecular condensate" specifically refers to a localized concentration of molecules, which should not be confused with "aggregation," a term that denotes a different process. Here, "condensation" indicates that molecules are densely packed within a particular area. These densely concentrated molecules can serve as scaffolds, enabling interactions among various other molecules within this concentrated environment in the cytosol, despite the absence of a surrounding membrane. The red regions illustrated in the referenced material signify interactions occurring between molecules within these condensates. These interactions are classified as "weak interactions" due to their transient nature, which distinguishes them from stronger bonds, such as covalent bonds. Weak 6 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 interactions promote dynamic and temporary associations, allowing molecules to participate in a range of processes without being permanently bound to one another. This process is highly dynamic, indicating that molecules continuously interact and move. It is crucial to recognize that molecules are never stationary; they are always in motion. In this context, "client" molecules refer to other molecules that are attracted to the condensate due to affinity or specific binding interactions. These client molecules bind to the biomolecular condensate, enabling their participation in various cellular functions. To illustrate this concept more clearly, consider the example of the nucleolus. While this may be mentioned in your textbook, it serves as an exemplary case among many others. The nucleolus is a biomolecular condensate composed of pre-mRNA, pre-ribosomal RNA, and proteins that ultimately mature to form ribosomes. These ribosomes subsequently exit the nucleolus and migrate to the cytosol to carry out protein synthesis. In this context, pre-ribosomal RNA is transcribed from ribosomal genes situated on the chromatin scaffold within the nucleolus. The maturation of these RNA molecules is facilitated by small nuclear RNA (snRNA) and other factors. This dynamic interaction makes the nucleolus an excellent representation of a biomolecular condensate, where various molecules, including RNA and proteins, are concentrated and interact dynamically within a specific cellular space without necessitating a surrounding membrane. This phenomenon contrasts with stable interactions, which involve more fixed structures. For instance, recall the image of the plasma membrane discussed previously, which depicted clusters of proteins and lipids forming microdomains. While these regions could be classified as "condensates," they differ from biomolecular condensates due to their reliance on stable interactions, such as covalent bonds. These stable structures do not exhibit the same dynamic changes as biomolecular condensates, which depend on weak, transient interactions, allowing them to continuously alter their shape and composition. Previously, it was noted that cellular compartments are organized based on their topological similarities, and they can be categorized into three primary groups. One such category is the endolysosomal system, which facilitates communication and transport through vesicular trafficking. This process involves the exchange of vesicle membranes among various organelles, including the endoplasmic reticulum (ER), Golgi apparatus, and the entire endolysosomal system, dedicated to forming and regulating vesicular transport functions. The second category, gated transport, regulates the movement of proteins and other molecules between the cytosol and the nucleus. This process is vital for a variety of cellular functions. The third category discussed was protein translocation, particularly concerning organelles such as mitochondria, the endoplasmic reticulum, and peroxisomes. This process encompasses more than just protein translocation; it also includes vesicular trafficking, as indicated by the green labeling in the notes. As previously stated, compartments such as the ER and Golgi apparatus receive materials through both protein translocation and vesicular trafficking. Additionally, the concept of membrane contact sites was introduced. These are specific regions where the membranes of different organelles, such as mitochondria and ER or mitochondria and lysosomes, come into close proximity without fusing. This close arrangement facilitates the exchange of lipids, ions, and various regulatory molecules essential for signaling, metabolism, and overall cellular responses. Lastly, the topic of engulfment was discussed, particularly in the context of autophagy. Engulfment refers to the process by which cellular components are encircled by a membrane to form an autophagosome. This membrane typically originates from other sources, such as the endoplasmic reticulum or, in some instances, the mitochondria. Ultimately, the autophagosome fuses with lysosomes, leading to the degradation of its contents through lysosomal-mediated pathways. This process is crucial for recycling cellular components and maintaining cellular health. The principles of vesicle transport warrant careful examination, particularly the regulated mechanisms governing its bioenergetic parameters of time and space. A fundamental aspect of vesicle transport regulation is membrane polarization. Membranes exhibit asymmetry, a feature observed in both plasma membranes and the internal membranes surrounding organelles. For example, when an exocytic vesicle fuses with the plasma membrane, its lumen contents 7 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 become exposed to the extracellular environment. This orientation dictates that any sugars attached to proteins on the vesicle's surface will face the extracellular matrix. In vesicular transport between organelles, membrane orientation is crucial. During vesicle formation, the lumen of the vesicle aligns with that of the target organelle, ensuring consistent polarization—particularly in the insertion of transmembrane proteins—throughout the transport process. This regulation is essential due to the distinct environments within organelles compared to the cytosol; for instance, pH levels within organelles can differ markedly from those in the cytosolic environment, influencing various biochemical reactions and molecular interactions. Each organelle maintains a distinct pH compared to the cytosol, a variation critical to its specialized functions. These pH differences result from varied ion concentrations within each compartment; for instance, mitochondria and lysosomes exhibit specific pH levels essential for functions such as calcium storage and cellular signaling. Protein transport into these compartments is tightly regulated through signaling mechanisms. Proteins requiring translocation into organelles like mitochondria or the endoplasmic reticulum (ER) contain specific signal sequences that facilitate their import. These sequences often consist of particular amino acid stretches—either hydrophobic, hydrophilic, or a combination thereof—creating unique signatures for each protein. For example, proteins targeted for import into the ER generally possess hydrophobic amino acid sequences. These regions are recognized by the translocon complex in the ER membrane, enabling proper insertion into the membrane or folding within the ER lumen. Similarly, proteins destined for organelles such as mitochondria or peroxisomes carry distinct signal sequences that guide their transport. These signals ensure accurate targeting, whether for nuclear import or export, thereby supporting the specialized functions of each cellular compartment. This precise regulation of protein transport is fundamental to cellular homeostasis and the overall functionality of the cell. Protein translocation: the endoplasmic reticulum The endoplasmic reticulum (ER) plays a central role in protein synthesis and processing, with protein import into the ER classified primarily by two methods: co-translational and post-translational transport. Co-translational transport involves the synthesis of proteins directly at the ER membrane. During this process, ribosomes attach to the ER, and as they translate mRNA into a polypeptide, the emerging protein is threaded directly into the ER lumen or integrated into the ER membrane. This method allows for seamless transfer of the nascent polypeptide into the ER as it is synthesized. In contrast, post-translational transport applies to proteins fully synthesized in the cytosol before their import into the ER. For such proteins, entry into the ER requires unfolding to facilitate their passage through the ER membrane. Both co-translational and post-translational pathways require precise regulation to ensure correct protein targeting to the ER. Proteins destined for the ER typically contain a specific signal sequence, often composed of hydrophobic amino acids, which serves as a recognition marker for import. This signal is identified by a receptor in the ER membrane. The signal recognition particle (SRP) is a critical intermediary in this process, particularly for co-translational transport. The SRP binds to the signal sequence on the nascent protein, temporarily halting translation until the ribosome docks with the ER membrane. This pause ensures that the protein is accurately directed and integrated into the ER, optimizing the efficiency and precision of cellular protein transport and processing mechanisms. The signal recognition particle (SRP) is classified as a ribonucleoprotein, indicating it is a complex composed of both protein and RNA elements. In this context, the RNA component is neither structural nor a messenger RNA; rather, it functions catalytically, facilitating the transport of proteins into the endoplasmic reticulum (ER). The SRP's structure allows it to selectively recognize and bind to the hydrophobic amino acid sequence on the nascent protein that designates it for ER import. 8 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 Beyond the SRP, two additional components are essential for co-translational protein translocation: the ER membrane receptor and the ribosome. Together, these elements coordinate the protein's seamless transfer into the ER. The process unfolds as follows: as the ribosome synthesizes the nascent protein, the SRP binds to it, temporarily pausing translation. This pause continues until the ribosome docks with the ER membrane via the receptor, establishing a cycle among the SRP, receptor, and ribosome. Once correctly positioned, the nascent protein is translocated through a structure called the translocon, a channel in the ER membrane that facilitates the protein’s entry into the ER as it is synthesized. This co-translational translocation is a highly dynamic process, with translation and import occurring simultaneously. Tight regulation of this mechanism, through coordinated interactions among the SRP, receptor, and ribosome, ensures accurate delivery of proteins into the ER, where they undergo proper folding and maturation. What are polyribosomes? Polyribosomes, or polysomes, are clusters of ribosomes that translate a single mRNA molecule simultaneously, facilitating the rapid and efficient synthesis of multiple copies of a protein. In the context of co-translational import into the endoplasmic reticulum (ER), proteins synthesized on these polysomes can be categorized as either soluble proteins that localize within the ER lumen or as transmembrane proteins integrated into the ER membrane. Proteins destined for the ER include both resident ER proteins and those targeted for further transport, such as to the Golgi apparatus. Soluble proteins acquire their final conformation within the ER lumen, while transmembrane proteins contain hydrophobic sequences that signal their insertion into the ER membrane, enabling them to anchor as integral membrane proteins. Once in the ER, proteins are sorted based on their cellular destination: resident ER proteins remain in the ER (either within the lumen or membrane), while others are transported to the Golgi apparatus for further modifications. Following processing in the Golgi, proteins may be directed to lysosomes, incorporated into the plasma membrane, or secreted out of the cell via exocytosis, ensuring precise localization essential for cellular function. (See Alberts’ Molecular Biology of the Cell, Ch. 12) Post-translational protein import refers to the transport of proteins into cellular compartments after synthesis is complete, but before the protein has fully matured. For effective import, the protein must remain in an unfolded state, which is essential for its ability to traverse membranes. This unfolded state is maintained during synthesis, allowing the protein to remain flexible enough to pass through the membrane with the assistance of specific receptors and transporters. Chaperone proteins are integral to this process. These molecules help maintain proteins in their unfolded state during transport or, depending on the type of chaperone, assist in correct folding once the protein reaches its destination. Different classes of chaperones perform distinct functions across the cell. For post-translational import, chaperones stabilize the unfolded state to facilitate membrane translocation. This process is ATP-dependent, highlighting its active transport nature and the energy required for successful import. Additionally, compartmentalization within the endoplasmic reticulum (ER) supports various biochemical reactions, emphasizing the specialized environment needed for precise protein processing and maturation. Can anyone remind me of the types of reactions that take place there? In the endoplasmic reticulum (ER), several essential biochemical modifications take place, crucial for protein maturation and membrane asymmetry. One such modification is the attachment of glycosylphosphatidylinositol (GPI) anchors to proteins. GPI anchoring involves conjugating an amphipathic lipid, which contains a hydrophilic phosphate head, to a protein, allowing it to embed in the membrane with orientation toward the extracellular environment once it reaches the 9 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 plasma membrane. This anchoring occurs in the ER lumen, where GPI-modified proteins are correctly positioned for eventual exposure outside the cell. Beyond GPI anchoring, two other significant types of modifications occur in the ER: disulfide bond formation and glycosylation. Disulfide bonds are essential for stabilizing proteins, particularly those that will be extracellular. These bonds are formed through oxidation-reduction reactions, which are catalyzed by enzymes like protein disulfide isomerase (PDI), ensuring proper structural stability. Glycosylation is another critical ER modification that plays a role in protein folding and function. In the ER, the primary form of glycosylation is N-glycosylation, where sugars are attached to the nitrogen atom in the side chain of asparagine residues. This attachment involves complex sugars, including N-acetylglucosamine, mannose, and glucose units. These glycan structures act as molecular tags that monitor and regulate protein processing in the ER, helping ensure that each protein has been correctly synthesized and folded. To further aid in protein folding, chaperone proteins like calnexin are active in the ER. Calnexin specifically binds to partially folded glycoproteins, assisting in their proper conformation before they advance to subsequent processing stages. While glycosylation begins in the ER, further modification occurs in the Golgi apparatus, where additional glycosylation steps refine and finalize the glycan structures. These modifications serve functional roles, including protection from degradation, correct folding, and the precise targeting of proteins within or outside the cell. What is the origin of the name calnexin? The protein chaperone calnexin derives its name from its reliance on calcium ions and serves a critical function in quality control within the endoplasmic reticulum (ER), assessing protein folding accuracy. Initially, when a nascent protein undergoes glycosylation in the ER, it receives a glycan structure with three glucose residues. During the folding process, two of these glucose units are removed, leaving one glucose residue, which then binds to calnexin. This single glucose residue serves as an indicator that the protein is progressing towards a correctly folded conformation. This binding constitutes the first cycle of calnexin's interaction with the protein. If the protein achieves its correct conformation, it progresses to the next stages of cellular transport. However, if the protein remains misfolded, an ER retention receptor identifies this incorrect folding. In response, another chaperone binds to the protein, catalyzing the re-addition of a glucose molecule, thereby restarting the cycle with calnexin. This iterative process continues, with calnexin re-engaging until the protein ultimately attains its proper, functional structure. The Unfolded Protein Response (UPR) is a cellular response triggered by the accumulation of misfolded or aggregated proteins in the endoplasmic reticulum (ER). However, activation of the UPR depends on specific characteristics of the accumulating proteins. Unlike biomolecular condensates, which are organized and reversible, aggregation refers to the clumping of multiple misfolded proteins, often due to structural mutations that disrupt proper folding, resulting in a "gain of function" anomaly. When excessive or potentially toxic aggregates are detected, the cell may initiate the UPR as part of an effort to restore homeostasis. In addition to UPR activation, cells utilize other mechanisms to manage misfolded proteins, including their export from the ER and subsequent degradation by the proteasome. The proteasome is a large, stable protein complex that, unlike organelles, lacks a surrounding membrane. Misfolded proteins targeted for proteasomal degradation are first exported from the ER, a process requiring ATP, as they must be unfolded to exit the ER regardless of any prior incorrect folding. 10 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 Once unfolded, the misfolded proteins are tagged with polyubiquitin chains, signaling them for degradation by the proteasome. This ubiquitin-proteasome system ensures the selective clearance of defective or surplus proteins, thereby preventing potential cellular toxicity and supporting protein quality control. Polyubiquitin chains are formed by linking multiple ubiquitin molecules, each consisting of 76 amino acids, in various configurations tailored to specific regulatory roles. In the case of protein degradation, the polyubiquitin chain is recognized by the proteasome as a unified signal, directing it to degrade misfolded or excess proteins. However, it is important to clarify that the presence of misfolded proteins in the ER does not automatically trigger the Unfolded Protein Response (UPR). Activation of the UPR is dependent on factors such as the specific properties of the misfolded protein, its accumulation level in the ER, and the cell type involved. Certain cell types, like neurons and muscle cells, which are non-dividing, often exhibit heightened sensitivity to misfolded proteins, as they lack the ability to dilute accumulated proteins through cell division. In contrast, dividing cells may have a lower threshold for UPR activation, partially due to their ability to distribute the protein load across new cells during division. Consequently, tissue type and physiological context critically shape cellular responses to ER stress. These stress responses can be classified as adaptive or maladaptive. Adaptive responses are transient and help the cell manage temporary stress effectively, enabling a return to homeostasis. In contrast, maladaptive responses occur when stress becomes chronic, leading to prolonged activation of cellular pathways that do not resolve the issue and may, over time, contribute to cellular dysfunction or toxicity. This maladaptive response emerges when prolonged stress outpaces the cell’s coping mechanisms and is particularly detrimental in the context of continuous protein misfolding or aggregation. Thus, cellular outcomes in response to stress are not universally successful and are influenced by several factors, including the specific cell type, the characteristics of the misfolded protein, and ER conditions. Understanding these contexts is crucial, as they determine whether the cell achieves a beneficial adaptive response or shifts into a potentially harmful maladaptive state. The image above illustrates the three distinct branches of the Unfolded Protein Response (UPR), each of which plays a significant role in cellular stress management. Understanding these pathways is critical as they represent potential targets for therapeutic interventions aimed at enhancing the UPR to mitigate cellular stress. The three branches include: 1. IRE1 (Inositol-Requiring Enzyme 1): This pathway culminates in the production of molecular chaperones that facilitate proper protein folding within the endoplasmic reticulum (ER). 2. PERK (PKR-like ER Kinase): This branch reduces overall protein translation, thereby alleviating the burden on the ER during stress conditions. 3. ATF6 (Activating Transcription Factor 6): This pathway activates various responses to restore ER function and improve cellular adaptation to stress. The regulation of these branches and their interactions during cellular stress is complex. Cells may activate different branches of the UPR in response to specific stressors, and the balance between these pathways can vary considerably between cell types. Consequently, introducing pharmacological agents that target a specific branch of the UPR may not yield uniformly beneficial outcomes. Biological responses are modulated by numerous factors, including the timing of activation, spatial context, and the degree of upregulation or downregulation of each pathway. 11 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 When drugs are employed to correct dysfunctional processes, particularly those affected by mutations or disease states, they may disrupt the existing cellular equilibrium. This disruption complicates the prediction of therapeutic outcomes, highlighting the need for comprehensive evaluations of both safety and efficacy during preclinical and clinical trials. For instance, the first branch of the UPR, involving IRE1, promotes the synthesis of chaperones that assist in protein folding. In parallel, the second branch, mediated by PERK, phosphorylates eukaryotic elongation factor 2 (eEF2), thereby inhibiting protein translation to alleviate stress. Understanding these intricate mechanisms and their potential for intervention is essential for developing effective therapeutic strategies. The role of eEF2 The phosphorylation of eukaryotic elongation factor 2 (eEF2) plays a crucial role in the regulation of translation initiation. When eEF2 is phosphorylated, its ability to effectively bind to the Kozak sequence of mRNA is significantly reduced, which is essential for proper translation initiation. This phosphorylation event results in a downregulation of the overall translation process, limiting the production of new proteins within the cell. Importantly, translation does not cease entirely; instead, it is selectively attenuated. This selective attenuation allows for the continued synthesis of specific proteins, particularly chaperones, which are vital for managing cellular stress. Chaperones assist in the proper folding of proteins and help prevent aggregation, thereby playing a critical role in cellular responses to stress. In the context of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), neurons are subjected to stress from misfolded or aggregated proteins. This stress triggers the unfolded protein response (UPR), which serves to mitigate the effects of these problematic proteins. The UPR temporarily reduces global protein synthesis, thereby preventing the accumulation of misfolded proteins that could overwhelm the cellular machinery. A key mechanism of this translational repression involves the phosphorylation of eIF2-alpha. This modification decreases the overall rate of translation, limiting the production of new proteins. However, it is important to note that this inhibition is selective, allowing for the continued synthesis of essential proteins such as chaperones that assist in protein folding. The protein GADD34 plays a significant role in reversing eIF2-alpha phosphorylation, thereby resuming normal translation levels. The inhibition of GADD34 could prolong the phosphorylated state of eIF2-alpha, further extending the reduction in protein synthesis and aiding the cell in managing stress more effectively. Theoretical implications suggest that blocking GADD34 might provide neurons with additional time to process misfolded proteins. However, such an approach carries potential risks, as inhibiting GADD34 could lead to unpredictable effects in other tissues. Given its involvement in basic cellular functions, the inhibition of GADD34 may create a risk for side effects, underscoring the importance of carefully considering the broader implications of targeting this pathway in therapeutic contexts. Peroxisomes Peroxisomes are indeed remarkable and crucial organelles that often do not receive the attention they deserve in discussions of cellular biology. Their primary roles include the metabolism of fatty acids and the detoxification of harmful substances, both of which are essential for maintaining cellular health. Peroxisomes are small, membrane-bound organelles found in almost all eukaryotic cells. They are particularly abundant in the liver and kidney cells, where their detoxifying functions are vital. One of the hallmark features of peroxisomes is their ability to carry out oxidative reactions, which lead to the production of hydrogen peroxide (H₂O₂). While H₂O₂ is a reactive oxygen species that can be damaging to cells, peroxisomes contain the enzyme catalase, which efficiently converts hydrogen peroxide into water (H₂O) and oxygen (O₂), neutralizing its potential harmful effects. Functions of Peroxisomes 1. Fatty Acid Metabolism: Peroxisomes play a key role in the oxidation of very long-chain fatty acids (VLCFAs) into medium-chain fatty acids, which can then enter the mitochondria for further oxidation. This process is critical for the breakdown of fats and the production of energy. 2. Detoxification: Peroxisomes detoxify various harmful byproducts of metabolism. Similar to the smooth endoplasmic reticulum, they oxidize substances like amino acids and alcohols, generating hydrogen peroxide in the process. Catalase then acts on H₂O₂ to convert it into less harmful products, thereby protecting the cell from oxidative damage. Visualization of Peroxisomes 12 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 To study peroxisomes and other small organelles, scientists often employ advanced imaging techniques like electron microscopy. This method allows for high-resolution imaging, enabling researchers to observe structures at the nanometer scale. For instance, a scale bar indicating 200 nanometers emphasizes the small size of peroxisomes, allowing us to visualize individual organelles and even molecular structures. Peroxisomes appear highly electron-dense under microscopy due to their high concentration of enzymes, lipids, and metabolic substrates. This electron density is a direct reflection of their packed nature, which is critical for their metabolic activities. Peroxisomes are indeed multifunctional organelles that play vital roles not only in detoxification but also in biosynthesis, particularly in the metabolism of fatty acids and the production of essential lipids. Let's explore their functions and formation in greater detail. Biosynthesis and Metabolism in Peroxisomes 1. Beta-Oxidation: a. Peroxisomes are critical for the breakdown of very long-chain fatty acids (VLCFAs) through a process known as beta-oxidation. During this process, long fatty acid chains are transported into peroxisomes, where they undergo chemical breakdown. b. The enzymatic reactions in beta-oxidation cleave covalent bonds within the fatty acids, resulting in shorter-chain fatty acids and the production of acetyl-CoA. This acetyl-CoA can then enter various metabolic pathways, such as glycolysis or the Krebs cycle, contributing to cellular energy production. 2. Production of Plasmalogens: a. One of the intermediate products of beta-oxidation in peroxisomes is plasmalogens, a type of ether lipid that is essential for the integrity of cell membranes, particularly in myelin sheaths that surround neurons in both the central and peripheral nervous systems. b. Plasmalogens play a critical role in membrane fluidity and functionality, and their synthesis in peroxisomes underscores the organelle's importance in lipid metabolism and cell membrane composition. Formation of Peroxisomes Peroxisomes have a unique formation process compared to other organelles: 1. De Novo Formation: a. Unlike organelles such as mitochondria and the endoplasmic reticulum (ER), which expand from pre- existing structures, peroxisomes can form de novo. This means they can be generated from scratch through the fusion of smaller vesicles. b. This capability allows the cell to produce new peroxisomes as needed, adapting to varying metabolic demands and detoxification requirements. 2. Vesicle Fusion: a. The formation of peroxisomes primarily occurs through the fusion of vesicles that are derived from the ER. These vesicles contain enzymes and proteins essential for peroxisomal function. b. As these vesicles fuse, they build up the peroxisome, which can subsequently import additional proteins and fatty acids from the cytosol. Role of Peroxins (PEX Proteins) The import of proteins and fatty acids into peroxisomes is facilitated by specific transport proteins known as peroxins (PEX proteins), such as PEX1, PEX2, PEX7, and PEX9. These proteins are crucial for recognizing and transporting essential fatty acids and enzymes into the peroxisome. Notably, proteins imported into the peroxisome must be unfolded to pass through the membrane, while those embedded in the membrane typically originate from ER-derived vesicles. Peroxisomal Dysfunction When peroxins are mutated or dysfunctional, it can severely impact peroxisome function. This can result in the accumulation of long-chain fatty acids in the cell, leading to toxic effects and the inability to produce essential lipids like plasmalogens. Such malfunctions are linked to various metabolic disorders, including Zellweger syndrome, where peroxisomal biogenesis is impaired, leading to severe developmental and metabolic issues. 13 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 Disorders like Zellweger syndrome and X-linked adrenoleukodystrophy arise from defects in peroxisomal function, leading to an accumulation of VLCFAs and other toxic metabolites in the body. This highlights the importance of peroxisomes in maintaining metabolic balance and detoxification processes. In summary, peroxisomes are essential organelles that not only detoxify harmful substances but also play a key role in lipid metabolism and biosynthesis. Their efficient functioning is crucial for cellular health, and understanding their structure and function offers insights into various metabolic disorders associated with their dysfunction. Their unique formation process through vesicle fusion, combined with the critical functions of peroxins, underscores their adaptability and importance in cellular health. Understanding peroxisomes' roles and how they form can provide insights into metabolic disorders and potential therapeutic interventions. A severe consequence of such mutations is Zellweger syndrome, a congenital disorder characterized by very low or absent myelin in the brain, which is evident in MRI scans. This leads to severe neurological issues, as well as abnormalities in organs like the liver and kidneys, which rely heavily on fatty acid metabolism. Zellweger syndrome is typically fatal in early infancy due to the body’s inability to process long-chain fatty acids and maintain proper cellular membranes. Protein translocation: mitochondria Mitochondria are indeed remarkable organelles, playing a pivotal role in cellular energy production and metabolism. Their functionality relies heavily on the precise import of proteins from the cytosol. Let’s break down the processes involved in mitochondrial protein import, the significance of this mechanism, and the interplay between various transport complexes. Why do mitochondria need to import proteins? 1. Limited Genetic Capacity: a. Despite having their own circular DNA, mitochondria can only encode a limited number of proteins— primarily tRNAs, ribosomal RNAs, and a few essential proteins. This limited genome is insufficient for all the proteins required for mitochondrial functions. b. Many proteins necessary for critical processes like oxidative phosphorylation, which occurs in the cristae, must be synthesized in the cytosol and then imported into the mitochondria. 2. Energy Production and Metabolism: a. Mitochondria are known as the "powerhouses of the cell" because they generate ATP through oxidative phosphorylation. This process relies on a specific set of proteins that must be imported from the cytosol. b. Proper functioning of these proteins is crucial for maintaining the energy balance of the cell, making the import process essential for cell viability. Structure of Mitochondria Mitochondria have a double membrane structure that consists of: Outer Membrane: Contains porins that allow small molecules to pass through. Inner Membrane: Folded into cristae, where the machinery for oxidative phosphorylation is located. This membrane is impermeable to most ions and small molecules. Matrix: The innermost compartment containing enzymes for the Krebs cycle and the mitochondrial DNA. Each of these compartments has distinct functions, and proteins must be accurately directed to their appropriate locations. 14 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 Mechanisms of Protein Import 1. Signal Sequences: Proteins destined for mitochondria are tagged with a signal sequence, a specific stretch of amino acids that identifies them for import. This sequence is recognized by receptors on the mitochondria. 2. Translocator Complexes: Mitochondria utilize specialized translocator complexes for protein import: TOM (Translocase of the Outer Membrane): The first complex to interact with incoming proteins. It recognizes the signal sequence and facilitates the entry of unfolded proteins into the outer membrane. TIM (Translocase of the Inner Membrane): Transfers proteins from the TOM complex into the inner membrane or matrix. Depending on the destination within the mitochondria, TIM has sub-complexes like TIM22 (for inner membrane proteins) and TIM23 (for matrix proteins). OXA (Oxidase Assembly): Also assists in the insertion of proteins into the inner membrane, specifically those synthesized within the mitochondria itself. 3. Energy Requirements: The process of protein import is energy-dependent, primarily requiring ATP to unfold the incoming proteins and assist in their translocation across the membranes. Additionally, the membrane potential across the inner membrane contributes to this process. In order, the process of protein import in mitochondria can be summarized as follows: 1. Recognition and Binding: The signal sequence on the incoming protein is recognized by receptors on the TOM complex. The protein is then bound and transported through the TOM complex into the intermembrane space. 2. Transfer to TIM: Once in the intermembrane space, the protein is transferred to the TIM complex. Depending on its destination, the protein is directed to either the matrix or the inner membrane. 3. Final Targeting: If the protein is destined for the matrix, it is fully imported into the matrix. If it needs to be integrated into the inner membrane (such as for components of the electron transport chain), it will be processed through either the TIM22 or OXA complexes. Regulation of the Import Process The import process is highly regulated to ensure that only specific proteins with the appropriate signal sequences are imported. This prevents non-target proteins from entering the mitochondria, which could disrupt mitochondrial function. The TOM, TIM, and OXA complexes work together in a coordinated manner, particularly at the contact sites between the outer and inner membranes, ensuring proteins reach their designated compartments without error. In summary, mitochondria import proteins extensively due to their limited genetic capacity and the need for numerous specialized proteins to carry out essential metabolic functions. The intricate system of signal sequences and translocator complexes ensures that proteins are selectively and efficiently directed to their proper mitochondrial destinations. This precision is crucial for maintaining mitochondrial function, energy production, and overall cellular health. Disruptions in this import process can lead to mitochondrial dysfunction and various metabolic disorders, emphasizing the importance of understanding these mechanisms in cell biology. This section will address the energy requirements associated with cellular processes. Any biochemical activity that entails the formation of covalent bonds or phosphorylation necessitates an energy source. In this context, the transport mechanism is classified as active transport, indicating that it requires energy expenditure. The primary source of this energy is typically adenosine triphosphate (ATP). The hydrolysis of ATP results in the formation of adenosine diphosphate (ADP) and the release of inorganic phosphate. Additionally, transport mechanisms may utilize the membrane potential as an energy source. 15 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 It is important to recognize that mitochondria possess a membrane potential, as do all organelles within the cell. Specifically, the plasma membrane is another notable structure that exhibits a membrane potential. The differences in ion distribution between the intracellular and extracellular environments create this membrane potential, which exemplifies cellular asymmetry. The interior of the cell is negatively charged, primarily due to the presence of negatively charged ions and phosphatidylserine, which is located in the inner leaflet of the plasma membrane. While all organelles demonstrate some level of asymmetry, it should be noted that not all of them possess a significant membrane potential. In contrast, the inner membrane of mitochondria has a considerably higher membrane potential, typically ranging from 100 to 130 mV. This elevated potential results from the activities occurring in the intermembrane space during oxidative phosphorylation, where protons are pumped into this space, thereby establishing a charge differential. This electrochemical gradient is essential, as it provides the energy required for various transport processes. The membrane potential acts as an energy source, facilitating the import of essential substances into the mitochondria via transporters. Furthermore, oxidative and reductive reactions are coupled with these transport processes, enhancing the transfer of energy necessary for transport functions. Gated regulated transport: the nucleus Gated regulated transport is a crucial mechanism for maintaining the functional exchange of materials between the nucleus and the cytosol. Various types of materials are transported across the nuclear envelope. For instance, histones are transported from the cytosol into the nucleus, while RNA is transported from the nucleus to the cytosol. In addition to histones and RNA, numerous other essential materials must traverse the nuclear envelope, including proteins such as polymerases, which are vital for DNA replication and transcription, as well as splicing factors, enzymes, and various metabolites involved in nucleic acid metabolism. Another critical component is nucleotides, which serve as both an energy source and building blocks for nucleic acids. Shifting focus to larger structures, biomolecular condensates are also relevant. A prime example of a biomolecular condensate within the nucleus is the nucleolus, where ribosomal RNA (rRNA) and ribosomal proteins are assembled into ribosomes. Following assembly, these ribosomes must exit the nucleus and enter the cytosol to perform their functions. The transport of ribosomes necessitates the movement of substantial complexes through nuclear pores, indicating that these pores must be sufficiently large to accommodate the intricate assemblies of rRNA and proteins. Nuclear pores are integral to this transport process, connecting the inner and outer membranes of the nucleus and facilitating the exchange of large molecular assemblies. These pores are constructed from proteins known as nucleoporins, which, while diverse, assemble in large quantities to form a structure resembling a complex gate with a mesh-like quality. The functional mechanism of this structure is based on the presence of unstructured regions within nucleoporins, which impart a flexible, fibrillar appearance. This flexibility allows for the formation of a mesh that aids in transport, as these regions are rich in specific amino acids, particularly phenylalanine and glycine, enabling nucleoporins to accommodate various cargoes, including proteins requiring import from the cytosol into the nucleus. Electron microscopy provides insight into the arrangement of nucleoporins, revealing a rosette-like structure when viewed from above, with pores at the center and various protruding fibrils. The transport process through these pores involves the recognition of specific signals on cargo proteins by nuclear import receptors, which guide them through the nucleoporin mesh. Different receptors can recognize multiple cargo types, navigating through this structure in a highly regulated manner. 16 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 This transport is energy-dependent; however, the energy utilized is derived not from ATP but from the action of a small GTPase, which supplies the necessary driving force for regulated, directional movement through the nuclear pore. Small GTPases are pivotal in cellular regulation, particularly in the context of trafficking. A notable example includes the GTPases Rho, CDC42, and RAC. These proteins exist in two states: an active GTP-bound state and an inactive GDP- bound state. The transition between these states occurs through the hydrolysis of bound GTP, which converts it to GDP. Reactivation of the GTPase involves the exchange of GDP for GTP, a process facilitated by Guanine nucleotide Exchange Factors (GEFs). One illustrative example is the GTPase Ran. Within the nucleus, chromatin scaffolds support a protein known as Ran GEF, which catalyzes the exchange of GDP for GTP on Ran. Initially, Ran enters the nucleus in its inactive GDP-bound form. However, upon interaction with Ran GEF, it releases GDP and binds GTP, thus becoming active. This active form of Ran GTP then interacts with cargo molecules, allowing them to transit through the nuclear pore complex. Once Ran GTP is in the cytosol, it undergoes hydrolysis, resulting in the release of one phosphate group and converting back to the inactive Ran GDP form. This cyclical process maintains a concentration gradient, with Ran GDP predominantly in the cytosol and Ran GTP primarily within the nucleus. This gradient is crucial for regulating the directional movement of cargo into and out of the nucleus, thereby sustaining the transport balance within the cell. Previously the necessity of transporting various components such as mRNA, ribosomes, transcription factors, polymerases, and other essential proteins involved in DNA replication, transcription, translation, and RNA maturation 17 Molecular Cell Biology – Bolino Lesson 02 11th October 2024 was discussed. However, nuclear pores, often referred to as "gates," serve a purpose beyond facilitating this bidirectional transport of materials between the nucleus and the cytosol. They also play a critical role in regulating gene transcription and nucleotide function within the nucleus. An illustrative example of this regulation is calcineurin. In contrast to calnexin, calcineurin functions as a phosphatase that is activated by calcium. Once activated, calcineurin dephosphorylates specific substrates, including a transcription factor known as NFAT (or NFAT-C4, depending on the cell type). This transcription factor acts as a master regulator of various gene transcription programs. When phosphorylated, NFAT-C4 remains inactive in the cytosol. However, following activation by calcineurin, the phosphate groups are removed, resulting in the activation of NFAT-C4, which can then translocate to the nucleus and initiate downstream gene transcription. This selective gating mechanism enables cells to compartmentalize certain proteins, maintaining them in an inactive state in the cytosol until they are required in the nucleus. Thus, nuclear pores not only regulate the flow of necessary components but also control the timing and location of protein activity within the nucleus. This ensures that transcription factors and other regulatory proteins become active only when and where they are needed, thereby adding an additional layer of control to cellular function. 18 Molecular Cell Biology – Bolino Lesson 03 15th October 2024 Membrane trafficking and its regulation Vesicular transport The focus of this discussion is on vesicular transport that occurs through compartments with topological similarities. This transport involves movement between the plasma membrane and the extracellular environment, facilitating cellular communication with the surroundings. Additionally, attention will be directed to membrane contact sites, lysosomal function, and the role of the autophagosome in autophagy. The term "topologically similar" indicates that these compartments are structurally compatible for fusion in vesicular trafficking. In this process, transport occurs from a donor organelle or vesicle to a recipient compartment in a polarized, regulated manner. Polarization in this context refers to the directional preservation of vesicular contents and membrane orientation during transfer, ensuring that internal and external orientations are maintained in the new organelle. There are various types of vesicular transport: Exocytic transport: In this pathway, vesicles originate from the endoplasmic reticulum, progress to the Golgi apparatus, and then move to the plasma membrane in a highly regulated sequence. Fusion with the membrane requires sustained stimulation. Exocytosis also includes transport from the trans- Golgi network to the early endosome or from the Golgi to the late endosome and lysosome. Thus, exocytosis facilitates not only extracellular release but also intracellular trafficking. Endocytic transport: This pathway involves the internalization of molecules through endocytic vesicles derived from the plasma membrane, which are then directed to the early endosome. Following the endocytic route, these vesicles advance from the early endosome to multi-vesicular bodies, the late endosome, and ultimately the lysosome. Retrieval transport: This pathway recycles proteins and receptors, linking the plasma membrane and portions of the endosome through vesicles that return to the membrane. Additional retrieval routes connect the early endosome, lysosome, and late endosome back to the Golgi apparatus and, ultimately, the endoplasmic reticulum. Each of these pathways serves distinct cellular functions. These processes require precise regulation to ensure that each component reaches its designated destination. Various molecular elements play essential roles within these pathways, guiding each vesicle on its specific route and function. Each membrane is distinguished by a unique "identity" coating, composed of phosphoinositide-phospholipids (PIPs) and other associated proteins such as signaling molecules, GTPases, and phosphoinositide regulators. These components form specialized nanodomains on each membrane, giving them distinct molecular signatures. The donor vesicles are directed to target (acceptor) compartments, which they identify as compatible due to their similar molecular profiles. Vesicle transport occurs along the cytoskeletal network, primarily via microtubules. To maintain the distinct identities of donor and acceptor membranes, specific regulatory proteins are activated with each vesicular transfer. This transport activity is nearly continuous, with thousands of vesicles moving simultaneously throughout the cell, resulting in the constant internalization and redistribution of vesicular cargo. Phosphoinositide These signaling molecules exhibit highly dynamic behavior, converting between forms based on their specific functions and the membrane identity they encode. Anchored to the membrane by their fatty acid chains, these molecules extend into the cytosol to facilitate recognition and communication with acceptor membranes, presenting a hydrophilic side for such interactions. Phosphoinositides (PIPs) are derived through specific polyphosphorylation at the 3, 4, or 5 positions of phosphatidylinositol, their precursor molecule. This precise modification process enables PIPs to act as signaling lipids, changing in a controlled manner that effectors can recognize and 19 Molecular Cell Biology – Bolino Lesson 03 15th October 2024 bind. Protein effectors contain specialized domains that selectively recognize these lipids, ensuring accurate interactions based on the lipid composition of each membrane. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is localized primarily on the plasma membrane, where it serves as a major signaling lipid. PIP3 orchestrates downstream signaling of tyrosine kinase receptors, impacting cellular processes such as transcriptional regulation and cell cycle progression. Notably, PIP3 is concentrated at low levels within specific domains of the plasma membrane, precisely where tyrosine kinase receptors are present. This spatial restriction ensures that downstream signaling is focused within designated regions of the cell, thereby enhancing signal specificity. PIP3 also facilitates cytoskeletal remodeling, allowing the plasma membrane to undergo deformations necessary to accommodate pathogens. In addition, phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), another critical phosphoinositide, plays an essential role in the process of endocytosis. This regulatory process is further supported by vesicle- coating proteins, which aid in membrane recognition, deformation, and the selective transport of cargo. Membrane fusion is thus not solely for increasing surface area but is also a controlled mechanism for internalizing and trafficking cellular materials. Different types of coating proteins correspond to specific vesicular pathways: COPI proteins are involved in transport between the cisternae of the Golgi apparatus, assisting in vesicle formation and cargo selection. COPII proteins mediate transport from the endoplasmic reticulum (ER) to the Golgi, facilitating both membrane formation and cargo selection for this pathway. Additionally, clathrin plays a central role in vesicular trafficking. Clathrin is composed of a heavy and a light chain; the light chain interacts with actin filaments, while the heavy chain, through adaptors, selects cargo for transport. Beyond its role in clathrin-mediated endocytosis, clathrin is also essential for the transport of lysosomal enzymes from the Golgi to the lysosome. Clathrin assembles into a triskelion structure that supports membrane deformation in coordination with actin and enables precise cargo selection. Receptors are essential for the selective internalization of molecules, which cannot freely cross the membrane. By binding specific ligands, receptors enable the targeted uptake of these molecules. Additionally, receptor-mediated endocytosis serves as a mechanism for signal downregulation; receptors are internalized along with their ligands, providing a rapid means to reduce signaling activity—more efficient than suppressing transcription of the receptor gene itself. In clathrin-mediated endocytosis, clathrin interacts with adaptor protein AP2, which aids in selecting cargo and assists in membrane deformation during vesicle formation. As the membrane begins to cluster around the cargo, AP2 supports clathrin in bending the membrane. For vesicle release, the membrane is severed with the aid of dynamin, an essential GTPase that facilitates the final scission of the vesicle. Once in the cytosol, the vesicle adopts a spherical shape due to exposure to the aqueous environment. Following vesicle formation, the clathrin coat is removed to expose underlying molecular signals, including phosphoinositides like PI(4,5)P2 and associated proteins. This uncoating process is critical, as the clathrin mask must be removed to reveal the vesicle’s molecular identifiers, guiding it to its correct intracellular destination. Membrane deformation during vesicle formation is facilitated by the coordinated actions of actin, clathrin, and BAR (Bin/Amphiphysin/Rvs) domain proteins. BAR proteins, which are alpha-helical and positively charged, exhibit high 20 Molecular Cell Biology – Bolino Lesson 03 15th October 2024 affinity for the negatively charged phospholipids in the membrane. This electrostatic attraction allows BAR proteins to bind tightly to the membrane surface, contributing to the curvature and stabilization required for vesicle budding. Clathrin and actin further support this process by generating and maintaining the necessary structural tension, enabling efficient membrane invagination and vesicle formation. The Arp2/3 complex plays a key role in directing actin polymerization by binding to existing actin filaments and initiating the formation of new filaments at specific angles, allowing for the rearrangement of the actin network. This mechanism supports membrane invagination by generating a directional force for vesicle formation. Additionally, actin polymerization occurs at the edge of the forming vesicle, where actin filaments extend to provide the mechanical push required to propel the vesicle outward toward the cytosol. This coordinated actin activity contributes significantly to the force needed for efficient vesicle budding and release. Dynamin functions as a constrictor protein during vesicle formation. Upon dimerization and GTP hydrolysis, dynamin undergoes a conformational change that physically tightens around the membrane neck, bringing the two membrane layers into close proximity and ultimately fusing them to release the vesicle. The GTPase activity, along with GTP hydrolysis, allows dynamin to assemble into ultrastructures that exert the force necessary to constrict and merge the membrane layers. Other coating proteins, including COPI and COPII, also mediate vesicular transport but differ in their regulatory mechanisms. In clathrin-mediated endocytosis, specific phosphoinositides serve as triggers, recruiting the adaptor protein AP2, the receptor, and the associated vesicular machinery. Conversely, COPI and COPII vesicle formation is regulated by distinct GTPases, such as Arf, Sar, and Rab, which act as signaling molecules to direct vesicle assembly and cargo selection. COPII-coated vesicles, characterized by a double-layered coating, mediate transport from the endoplasmic reticulum (ER) to the Golgi apparatus. This process is regulated by the small GTPase Sar1, which alternates between active and inactive states depending on its GTP or GDP-bound form. Guanine nucleotide exchange factors (GEFs) activate Sar1 by promoting the exchange of GDP for GTP, while GTPase-activating proteins (GAPs) return Sar1 to its inactive state through GTP hydrolysis. Once activated, Sar1 initiates the recruitment of additional COPII components, including Sec23, Sec24, Sec13, and Sec31. These proteins assemble to form a structured cage around the vesicle, stabilizing it for transport from the ER to the Golgi. Sec23 and Sec24 are involved in cargo selection and membrane curvature, while Sec13 and Sec31 contribute to the outer coat structure, completing the vesicle’s double-layered coating essential for this transport pathway. In addition to the coating proteins, various regulators play crucial roles in directing vesicle trafficking. Phosphoinositide species are generated through phosphorylation, and their subsequent dephosphorylation by phosphatases converts them into different forms, contributing to membrane identity and signaling. Rab GTPases are essential for vesicle trafficking, working in conjunction with phosphoinositides (PIPs) and SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins to define membrane identity and facilitate accurate targeting of vesicles. Together, Rab proteins, PIPs, and SNAREs orchestrate the specific interactions 21 Molecular Cell Biology – Bolino Lesson 03 15th October 2024 needed for vesicle docking and fusion with their intended membranes, ensuring precise delivery of cargo throughout the cell. This cooperative mechanism is critical for maintaining cellular organization and function. The figure on the side, represents the Ras superfamily. (see Alberts) GTP and GDP interconversion is facilitated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), which promote their phosphorylation and dephosphorylation. Guanosine diphosphate (GDP) dissociation inhibitor (GDI) is a regulatory protein that binds to small GTPases in their GDP-bound state, helping to maintain their inactive form in the cytosol. GTP is essential for the integration of various signaling events throughout the cell, not limited to the plasma membrane. Among the small GTPases, Rheb, a member of the Ras superfamily, plays a critical role in converging signals onto the mechanistic target of rapamycin complex 1 (mTORC1), which is vital for cell growth and metabolism. Ras proteins are particularly significant in cancer biology due to their roles in regulating cell growth, proliferation, and gene transcription. Small Rho GTPases, such as those in the Rho family, assist in the dynamic modification of the cytoskeleton, impacting cell shape and motility. Rab and Arf proteins are key small GTPases that are particularly relevant to membrane trafficking, working in concert to establish and maintain membrane identity. Miro, a relatively recent addition to the GTPase family, is bound to the outer mitochondrial membrane and plays a role in mitochondrial transport and dynamics. While most of these GTPases are associated with various membranes, the exception is Run, which cycles in and out of the nuclear core in its GTP- and GDP-bound forms. In the nucleus, GTP interacts with GEF, which binds to chromatin at the nuclear lamina, indicating the involvement of GTPases in nuclear signaling processes as well. Rab Small GTPases, particularly Rab proteins, play a pivotal role in governing membrane fusion and are integral components of the vesicular coat that defines membrane identity. Rab proteins belong to the Ras superfamily and are essential for membrane trafficking, with each member designated by a numerical identifier that corresponds to its specific function and localization within the cell. For instance, Rab5 is primarily localized to early endosomes, while Rab7 is associated with late endosomes. These Rab proteins serve as important markers that guide vesicles to their appropriate organelle destinations. The fusion process is mediated by the activation of Rab proteins and SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Rab proteins recognize and interact with specific effector proteins, facilitating the recruitment of vSNARE (vesicular SNARE) and tSNARE (target SNARE) proteins. These cognate pairs of SNARE proteins bind together, bringing the opposing membranes into close proximity to promote fusion. To ensure that these membranes are effectively brought together, motor proteins facilitate the transport of vesicles along the cytoskeletal network, guiding them toward their target membranes and enhancing the likelihood of successful fusion. This intricate interplay between Rab proteins, SNAREs, and motor proteins is essential for efficient membrane trafficking and cellular communication. 22 Molecular Cell Biology – Bolino Lesson 03 15th October 2024 The formation of nanodomains responsible for membrane identity is a complex process involving phosphoinositides (PIPs), SNARE proteins, and Rab GTPases. This process is initiated by a key player that triggers a cascade of events leading to the assembly of the necessary protein components at a specific membrane location. To establish this platform, an initial Rab protein is activated by a guanine nucleotide exchange factor (GEF), facilitating the exchange of GDP for GTP, thereby converting Rab into its active form. This active Rab promotes the activity of a kinase that phosphorylates phosphatidylinositol, generating phosphatidylinositol (3,4,5)-trisphosphate (PIP3). The presence of PIP3 subsequently recruits additional GEFs that bind to the already active Rab, further stimulating the activation of more Rab molecules. This amplifies the signaling cascade, leading to the formation of microdomains that organize and concentrate essential proteins, beginning from a single activated molecule. This dynamic process highlights the intricate regulatory mechanisms that govern membrane identity and the coordination of signaling events critical for cellular function. Phosphoinositides (PIPs), SNARE proteins, and Rab GTPases collectively form the identity coat of membranes, enabling membrane fusion and defining specific cellular functions. As vesicles mature, they must acquire a new identity, a process that is distinct from initial fusion events. Early endosomes originate from vesicles that fuse with the plasma membrane via endocytosis. However, as they mature, they undergo significant changes that alter their identity. For instance, early endosomes can transition to become precursors of lysosomes. This transition involves the loss of Rab5 identity and the acquisition of Rab7, which marks the endosome's progression toward late endosome status. The conversion from Rab5 to Rab7 occurs through the activation of specific effectors that are required for the function of the membrane, as well as GEFs (guanine nucleotide exchange factors) that are particular to Rab7. As Rab5 is inactivated, the membrane increasingly expresses Rab7, signifying its transition to a late endosome. This maturation process is also associated with changes in phosphoinositide composition, contributing to the new identity of the membrane as it develops into a late endosome or lysosome. Thus, the dynamic interplay of Rab proteins and PIPs is critical for the maturation and functional specialization of endosomal compartments within the cell. Transport from the ER to Golgi COPII-coated vesicles are characterized by a double-layered coat that facilitates the transport of cargo from the endoplasmic reticulum (ER) to the Golgi apparatus. This double coating is assembled through the recruitment of various regulators to form macrodomains, which in turn attract additional proteins necessary for cargo selection and transport. The selection of cargo is a critical step, as proteins destined for the Golgi must be appropriately identified and packaged for transport. These proteins may undergo modifications such as glycosylation in the Golgi, or they may include enzymes and adaptors needed for specific functions in other cellular compartments. Therefore, it is essential to ensure that only correctly folded proteins are allowed to exit the ER. Proteins that are not correctly folded are assisted by chaperone proteins, which facilitate proper folding before transport. If a protein fails to achieve the correct conformation despite these chaperone interventions, it is targeted for degradation via the proteasomal pathway. This quality control mechanism ensures that only functional proteins are transported to their subsequent destinations, maintaining cellular integrity and function. COPII-coated vesicles bud from the endoplasmic reticulum (ER) and do not immediately fuse with the Golgi apparatus; instead, they first undergo maturation within a Golgi-associated compartment. During this maturation process, these vesicles acquire functions characteristic of other compartments, effectively switching their identity. 23 Molecular Cell Biology – Bolino Lesson 03 15th October 2024 The homotypic fusion of these vesicles is facilitated by the coordinated action of Rab GTPases and SNARE proteins. Both vSNAREs (vesicular SNAREs) and tSNAREs (target SNAREs) are present on the vesicles and the target membranes, respectively. These proteins interact to bring the membranes into close proximity, enabling fusion and the formation of vesicular tubular clusters. This process is crucial for the subsequent transport of cargo to the Golgi apparatus, allowing for the continuation of the vesicular trafficking pathway and the proper functioning of the secretory system within the cell. The vesicles involved in intracellular transport must remain associated with the cytoskeleton of the organelles they originate from. This attachment is crucial for maintaining the structural integrity and facilitating the movement of the vesicles within the cell. Several vesicles participate in the exchange of materials through COPI-coated transport, which is essential for recycling components such as receptors that are needed for future cellular functions. One critical sequence involved in this recycling process is the KDEL motif, a stretch of four amino acids found in proteins that reside within the endoplasmic reticulum (ER). Proteins containing the KDEL sequence are recognized by specific KDEL receptors that transport them from the Golgi apparatus back to the ER. Upon reaching the Golgi, these receptors undergo a cycle of transport through vesicles, ultimately facilitating the retrieval of KDEL-tagged proteins. This process is mediated by the interaction between the KDEL receptor and the C-terminal KDEL sequence of the proteins, ensuring that essential ER resident proteins are effectively recycled and retained within the ER for continued function. (See Alberts) There are several distinct types of glycosylation that occur within the cell, each playing a crucial role in protein function and localization: N-glycosylation involves the attachment of carbohydrate moieties to the nitrogen atom of asparagine residues within a protein. This modification serves as an important marker for assessing the correct folding of the protein, contributing to its stability and functionality. O-glycosylation refers to the covalent bonding of carbohydrates to the oxygen atom of serine or threonine residues. This type of glycosylation is characterized by the addition of sugar molecules to the lateral chains of these amino acids. During the maturation of proteins, multiple rounds of glycosylation occur. The initial glycosylation event begins in the endoplasmic reticulum (ER), where the mannose-6-phosphate marker is attached specifically to lysosomal enzymes. This modification is a critical post-translational modification that occurs in the Golgi apparatus and is unique to proteins destined for the lysosome. Subsequent modifications take place in the Golgi, including the removal of mannose residues and the addition of GlcNAc (N-acetylglucosamine) residues. These glycosylated proteins are then directed to their final destinations: they may either be transported to lysosomes via exocytosis or delivered to the plasma membrane through either regulated or unregulated pathways. This sophisticated process of glycosylation and subsequent trafficking is essential for the proper functioning of lysosomal enzymes and the overall homeostasis of the cell. (See Alberts for glycosylation types) Glycosylation is a multi-step process that begins in the endoplasmic reticulum (ER) and is essential for the proper folding and maturation of proteins. Initially, glucose residues are added to the nascent protein, and as folding progresses, these residues are sequentially removed until only a single glucose remains. The proteins that exit the ER typically have a high mannose content due to the addition of mannose residues during N-glycosylation. Upon leaving the ER, these glycoproteins undergo further modifications in the Golgi apparatus. Specifically, mannose residues are progressively removed, and N-acetylglucosamine (GlcNAc) is subsequently added to the protein structure. 24 Molecular Cell Biology – Bolino Lesson 03 15th October 2024 To assess the status of glycosylation, researchers can utilize Endo H, an enzyme that can differentiate between various glycosylation states of proteins. This enzyme is sensitive to the presence of high mannose structures; thus, if a protein is Endo H-sensitive, it indicates that the protein retains a high concentration of mannose and has not yet completed its maturation process within the Golgi. Conversely, if a protein is Endo H-resistant, it signifies that the protein has undergone further processing, having fewer mannose residues and more sialic acid, indicative of its presence in the later stages of maturation. This distinction can be monitored using techniques such as Western blot analysis, which allows for the visualization of glycosylation states. In addition to assisting in the proper folding and maturation of proteins, glycosylation also plays a crucial role in cellular signaling. Glycosylated proteins can serve as ligands for various receptors, facilitating important interactions that influence cellular communication and function. Transport from the trans-Golgi Exocytosis serves not only to deliver vesicles to the plasma membrane but also to transport enzymes to the lysosome. The tagging of lysosomal enzymes with mannose-6-phosphate (M6P) represents the initial reaction that occurs in the cis- Golgi. The lysosome contains approximately 70 different enzymes, each present in multiple copies, contributing to the organelle's compact yet highly dense structure. 1. Signal-mediated diversion to lysosome: The sorting of lysosomal enzymes occurs within the cis- Golgi, where exocytosis facilitates their delivery to lysosomes by conjugating the proteins with mannose-6-phosphate (M6P). Following this conjugation, selection is conducted with the assistance of clathrin, which aids in the identification of receptors for the M6P-tagged enzymes. Subsequently, vesicles containing these conjugated enzymes bud off from the membrane, during which the clathrin coat is removed. The resulting vesicles then fuse with lysosomes, which are formed through the fusion of multiple vesicles. In the lysosome, both the mannose-6- phosphate (M6P) receptor and the associated enzymes undergo disassembly due to the acidic pH unique to this organelle, which facilitates the dissociation of the receptor from its cargo. Following this process, the receptor is recycled, and depending on it