Molecular Cell Biology L2 Bolino PDF - 11 October 2024

Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 Cell compartments and protein sorting Cell compartments The key concept is that cells compartmentalize to ensure that materials reach the right place at the right time, facilitated by vario...

Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 Cell compartments and protein sorting Cell compartments The key concept is that cells compartmentalize to ensure that materials reach the right place at the right time, facilitated by various transport and localization systems. Why do cells have compartments? 1. To restrict specialized biochemical reactions by concentrating enzymes, substrates, regulators etc.; so it’s a way to concentrate the material needed. 2. To increase membrane area on which the reactions occur in many cases (e.g. oxidative phosphorylation). For example, mitochondria change shape and increase membrane surface area to meet cellular demands. However, while it enhances regulation, it complicates cellular processes because membranes are generally impermeable, which means that for substances like proteins or other necessary molecules to enter specific compartments or interact with membranes, there must be transport mechanisms in place. These mechanisms are essential because the membranes themselves don't naturally allow the passage of most substances. While this adds a layer of regulation and control, making the system more organized, it also introduces complexity. On one hand, it helps in maintaining specific internal environments, but on the other, it complicates the process, requiring specialized transport and regulatory systems. There are different intracellular compartments: 1. Nucleus and cytosol, connected through pore complexes 2. All organelles within the secretory and endocytic pathways including ER-Golgi-endo-lysosome- peroxisomes and connecting vesicles 3. Mitochondria So, these are three categories of compartmentalization within the cell. (She said she’s not going to explain what organelles are, but we need to refresh our minds with the Alberts). Fig.1 Fig.2 1 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 This tables show how much this compartmentalization occupies in relation to the total volume of the cell (Fig.1) or in relation as a percentage to the plasma membrane (fig.2). The relationship between cell volume and plasma membrane surface area is not directly proportional. Increasing the volume of a cell does not necessarily lead to a corresponding increase in the surface area of its plasma membrane. However, certain cells, like pancreatic cells and hepatocytes, have adapted by evolving a much larger proportion of membrane within their endoplasmic reticulum (ER), both smooth and rough, compared to their plasma membrane. This adaptation supports their specialized functions. Other types of cells, in contrast, have much less membrane in their ER. As we discussed before, sometimes what we observe in certain images, such as mitochondria in this case, might be misleading due to artifacts, not actual structures. Figure 3: example of electro-microscopy, organelles and stuff that are darker than others; if I have stuff that is denser, it’d be darker. This appearance is due to the fact that electron microscopy is typically performed on fixed cells. While electron microscopy is primarily used on fixed samples, the preparation and fixation techniques can vary significantly, allowing for improved image resolution. You might notice that some organelles, like lysosomes, appear darker in electron micrographs. Do you know why that is? In electron microscopy, denser structures within the cell appear darker. This is because the electron beam passes through the sample, and denser areas absorb or scatter more electrons, creating a darker image. Lysosomes, for example, appear dark because they are densely packed with a variety of enzymes—around seventy different enzymes, though not limited to just seventy—that are responsible for breaking down and metabolizing different substances. Out of these seventy enzymes, there are multiple copies in each lysosome, which makes them extremely dense, explaining why they appear darker than other cell structures in electron micrographs. In contrast, the cytoplasm appears lighter because it primarily contains water, ions, and other molecules, which are less dense. Cell compartments, or organelles, are typically surrounded by membranes, which is a fundamental concept in biology. You might be familiar with organelles like mitochondria, the nucleus, and the lysosomal system—all of which are membrane-bound. However, there are also biomolecular condensates, which are compartments not surrounded by membranes but still function as distinct cellular structures. This is a relatively new concept in biology, showing that not all cellular compartments require membranes to be functionally separate from the rest of the cell. Question: Why are they defined as compartments? 2 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 Because chemical reactions can occur within these biomolecular condensates. The term "biomolecular condensate" refers to a concentration of molecules in a specific space, not to be confused with "aggregation," which implies a different process. In this case, "condensation" means that molecules are more densely packed within a certain area. These molecules can act as scaffolds, allowing other molecules to interact and function within this concentrated environment in the cytosol, even though there's no membrane surrounding them. The red regions shown in your book represent interactions between molecules within these condensates. These are termed "weak interactions" because they are more transient than stronger bonds, such as covalent bonds. Weak interactions allow for dynamic, temporary associations, enabling the molecules to engage in various processes without being permanently bound. For example, this process is highly dynamic, meaning that molecules are constantly interacting and moving. It's important to remember that molecules are never stationary; they are always in motion. In this context, you also have "client" molecules, which are other molecules attracted to the condensate due to affinity or specific binding interactions. These client molecules bind to the biomolecular condensate, allowing them to participate in various cellular functions. To make this clearer and less abstract, let's look at an example: the nucleolus. Your textbook likely mentions this, but it's just one of many examples. The nucleolus is a biomolecular condensate made up of pre-mRNA, pre-ribosomal RNA, and proteins that, when mature, form ribosomes. These ribosomes will eventually leave the nucleolus and travel to the cytosol, where they carry out protein synthesis. In this process, pre-ribosomal RNA is transcribed from ribosomal genes located on the chromatin scaffold within the nucleolus. The maturation of these RNA molecules is aided by small nuclear RNA (snRNA), among other factors. This makes the nucleolus a perfect example of a biomolecular condensate, where many molecules, including RNA and proteins, are concentrated and interact dynamically within a specific cellular space without the need for a surrounding membrane. This is different from stable interactions, which involve more fixed structures. Do you remember the image of the plasma membrane we discussed last time? It showed clusters of proteins and lipids forming microdomains. While these regions could be called "condensates," they differ from biomolecular condensates because they are held together by stable interactions, such as covalent bonds. These stable structures don't change as dynamically as biomolecular condensates, which rely on weak, transient interactions, allowing them to continuously change shape and composition. Today, we discussed how cellular compartments are organized based on their topological similarities, and we can categorize them into three main groups. One of these categories is the endolysosomal system, which facilitates communication and transport through vesicular trafficking. This involves the exchange of vesicle membranes among various organelles, including the endoplasmic reticulum (ER), Golgi apparatus, and the entire endolysosomal system. This system is dedicated to forming and regulating functions related to vesicular transport. Next, we have gated transport, which regulates the movement of proteins and other molecules between the cytosol and the nucleus. This process is essential for various cellular functions. We also discussed protein translocation, particularly in relation to organelles like mitochondria, the endoplasmic reticulum, and peroxisomes. This process is not limited to just protein translocation; it also includes vesicular trafficking, as indicated by the green labeling in your notes. As previously mentioned, compartments such as the ER and Golgi apparatus receive materials through both protein translocation and vesicular trafficking. Furthermore, we introduced the concept of membrane contact sites, which we mentioned yesterday. These are specific areas where membranes from different organelles, such as the mitochondria and ER, or 3 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 mitochondria and lysosomes, come into close proximity without fusing. This proximity enables the exchange of lipids, ions, and various regulatory molecules that are crucial for signaling, metabolism, and overall cellular responses. Lastly, we talked about engulfment, particularly in the context of autophagy. Engulfment refers to the process where 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 cases, the mitochondria. The autophagosome ultimately fuses with lysosomes, leading to degradation of its contents through lysosomal- mediated pathways. This process is essential for recycling cellular components and maintaining cellular health. Let’s revisit the principles of vesicle transport that we discussed previously. As we mentioned, vesicle transport is a highly regulated process that operates within specific bioenergetic parameters of time and space. One of the primary regulations in vesicle transport is the polarization of membranes. Membranes are asymmetric, and this asymmetry applies to both plasma membranes and internal membranes surrounding organelles. For instance, consider an exocytic vesicle fusing with the plasma membrane: whatever is contained within the vesicle lumen is exposed to the outside environment. This means that any sugars attached to proteins on the vesicle’s surface will be oriented towards the extracellular matrix. When it comes to exchanging materials through vesicular transport between organelles, the process involves the orientation of the donor organelle's membrane. Specifically, the vesicle that is formed will have a lumen that corresponds to that of the target organelle. Importantly, the polarization—specifically, the way transmembrane proteins are inserted into the membrane—will remain consistent throughout this process. This regulation is crucial because the interactions that occur within the lumen of organelles differ significantly from those that happen outside in the cytosol. For example, the pH levels inside organelles can vary greatly from those in the cytosolic environment, which can impact the types of reactions and interactions that take place. 4 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 Each organelle has a different pH compared to the cytosol, and this variation is essential for their specific functions. The differences in pH arise because the ion concentrations are not the same in these compartments. For example, the mitochondria and lysosomes have distinct pH levels, which are crucial for their roles in processes like calcium storage and cellular signaling. Now, let's consider how proteins are transported into these compartments. Signaling plays a vital role in this regulation. Proteins that need to be translocated into organelles, such as mitochondria or the endoplasmic reticulum (ER), possess specific signals that facilitate their import. These signals often consist of stretches of amino acids that can be hydrophobic, hydrophilic, or a combination of both, creating a unique signature for each protein. For instance, to be imported into the ER, a protein typically contains a stretch of hydrophobic amino acids. This hydrophobic region is recognized by the translocon complex in the ER membrane, allowing the protein to be properly inserted into the membrane or folded into the lumen. In addition, proteins destined for organelles like the mitochondria or peroxisomes also have similar signal sequences that direct their transport. These sequences ensure that proteins are correctly targeted, either imported into or exported from the nucleus, thereby maintaining the specific functions of each compartment. This precise regulation of protein transport is crucial for cellular homeostasis and the overall functioning of the cell. Protein translocation: the endoplasmic reticulum Let's discuss the endoplasmic reticulum (ER) and how proteins are imported into it. The ER is crucial for protein synthesis and processing, and we can categorize the proteins that enter it based on two main methods: co-translational transport and post-translational transport. Co-translational transport occurs when ribosomes are attached to the membrane of the ER during protein synthesis. In this process, as the ribosome translates the mRNA into a polypeptide, the growing protein is threaded directly into the ER lumen or inserted into the ER membrane. In contrast, post-translational transport refers to proteins that are fully synthesized in the cytosol before being imported into the ER. In this case, the proteins need to be unfolded to cross the ER membrane, regardless of the method of transport. In both co-translational and post-translational transport, the delivery of proteins to the ER must be tightly regulated. Proteins destined for the ER typically contain a specific signal, often a highly hydrophobic amino acid stretch, that acts as a recognition signal for import. This signal is recognized by a receptor in the ER membrane. The receptor itself is regulated by another critical component called the signal recognition particle (SRP). The SRP binds to the signal sequence of the nascent protein and pauses translation until the ribosome is properly docked to the ER membrane. This ensures that the protein is correctly targeted and integrated into the ER, maintaining the overall efficiency and accuracy of cellular protein transport and processing. (rivedi Alberts) 5 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 The signal recognition particle (SRP) plays a crucial role as an intermediary that recognizes proteins destined for import into the endoplasmic reticulum (ER). The SRP binds to the signal sequence of the protein, which is being translated, facilitating its targeting to the ER. The term ribonucleoprotein describes the SRP, meaning it is a protein that associates with RNA. In this case, the RNA is not structural or messenger RNA; rather, it functions as a catalytic RNA, assisting in the protein's transport process. The structure of the SRP allows it to effectively recognize and bind to the hydrophobic amino acid stretch of the nascent protein that signals it for import. In addition to the SRP, there are two other key components involved in this process: the receptor in the ER membrane and the ribosome itself. Together, these components facilitate the co-translational translocation of the protein into the ER. Here’s how the process works: the SRP binds to the ribosome as it synthesizes the nascent protein, pausing translation until the ribosome is docked at the ER membrane. This forms a cycle between the SRP, the receptor, and the ribosome. Once the ribosome is correctly positioned, the nascent protein is translocated into the ER lumen through a translocon, which acts as a channel that allows the protein to enter the ER as it is being synthesized. This entire process is highly dynamic, with translation and import occurring simultaneously. The regulation of this process is tightly controlled by the interactions between the SRP, the receptor, and the ribosome, ensuring that proteins are accurately directed into the ER for proper folding and maturation. Question: What are polyribosomes? Polyribosomes, or polysomes, are clusters of ribosomes that simultaneously translate a single mRNA molecule, allowing for the efficient synthesis of multiple copies of a protein. When proteins are co-translated and imported into the endoplasmic reticulum (ER), they can either be soluble proteins that reside in the ER lumen or transmembrane proteins. Proteins destined for the ER include those that need to remain there and those that will be transported elsewhere, such as to the Golgi apparatus. Within the ER, soluble proteins adopt their final conformation while in the lumen, while transmembrane proteins possess hydrophobic stretches that serve as signals for their insertion into the ER membrane. These hydrophobic signals allow the proteins to integrate into the membrane and become anchored as transmembrane proteins. Once inside the ER, proteins can be categorized as resident proteins, which remain in the ER (either in the lumen or within the membrane), or those that must leave the ER for further processing. For example, proteins may be transported to the Golgi apparatus for post-translational modifications before being sent out of the cell via exocytosis or directed to lysosomes. ((I won't delve into these details right now, as they might be too complex. Instead, I encourage you to review this information in the slides and in Albert's textbook, particularly Chapter 12, in both the English and Italian versions)). We discussed that protein import can occur either co-translationally or post-translationally. When we refer to post-translational import, it means that the protein is not yet fully mature. Specifically, the protein must remain in an unfolded state for successful import. During synthesis, proteins are synthesized in a manner that allows them to maintain this unfolded conformation. This is crucial, as the unfolded protein must be able to pass through membranes, assisted by receptors and transporters. Chaperones play a vital role in this process. These proteins help maintain the unfolded state or assist in the correct folding of other proteins, depending on the specific chaperone involved. There are various classes of chaperones in the cell, each with distinct functions. In the case of post-translational import, chaperones help keep the protein unfolded so it can effectively traverse the membrane. It's important to note that this process is ATP-dependent, reflecting the active nature of the transport involved. (She’s summarizing quickly because this material can be found in the Alberts). 6 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 We also discussed the concept of compartmentalization in relation to various biochemical reactions that occur within the endoplasmic reticulum (ER). Question: Can anyone remind me of the types of reactions that take place there? As shown in the slides, several key reactions occur in the ER. One important process is the conjugation of GPI (glycosylphosphatidylinositol) anchors to proteins. This reaction is crucial for creating membrane asymmetry, as GPI anchors facilitate the attachment of proteins that need to be exposed on the extracellular side of the plasma membrane. Specifically, this involves the addition of an amphipathic lipid, which has a hydrophilic phosphate head, to the protein, allowing it to be properly positioned in the membrane. The process of GPI anchoring occurs in the lumen of the ER, enabling proteins to be correctly modified before reaching the plasma membrane. Once in the lumen, these proteins are indeed exposed to the extracellular environment when they arrive at the plasma membrane. In addition to GPI conjugation, several other important reactions take place in the ER. One notable reaction is the formation of disulfide bonds, which are essential for stabilizing proteins, particularly those that will be exposed to the extracellular environment. This stabilization involves oxidation-reduction reactions facilitated by enzymes such as protein disulfide isomerase. Another significant reaction occurring in the ER is glycosylation, which is vital for proper protein folding and function. We discussed glycolipids and glycoproteins, so where do these modifications occur? Initially, they take place in the endoplasmic reticulum (ER), but the Golgi apparatus is also a key site for glycosylation. The Golgi is primarily responsible for the extensive glycosylation of proteins, which serves various functional purposes. In the ER, the predominant type of glycosylation is N-glycosylation. To clarify the terminology: "N" and "O" refer to the type of amino acid residues involved—specifically, whether the sugar is attached to a nitrogen (asparagine) or an oxygen (serine or threonine) atom. In N-glycosylation, the sugar is covalently bound to the side chain of asparagine. This attachment involves complex sugars, not just simple monosaccharides. For instance, a typical N-glycan might include N-acetylglucosamine, several mannose residues, and glucose units. The specific structure of these sugar chains serves as a molecular tag that helps regulate the processing of the protein in the ER, indicating whether it has been correctly synthesized and folded. To assist in this folding process, a chaperone protein called calnexin plays a crucial role in the ER. Calnexin helps ensure that proteins achieve their proper conformation before they move on to further stages of processing. Q. Do you know why it's called calnexin? The name "calnexin" comes from its dependence on calcium. Calnexin plays a critical role in sensing whether a protein is correctly folded. Initially, when a protein receives its glycosylation, it has three glucose molecules attached. As the process unfolds, two glucose units are removed, leaving one glucose bound to calnexin. This binding indicates that the protein is well- formed and properly folded. This marks the first cycle of calnexin's interaction with the protein. If the protein is not correctly folded, the ER retention receptor recognizes this misfolded state. In such cases, another chaperone can bind to the protein, facilitating the addition of another glucose molecule to restart the cycle. This process continues until the protein achieves the correct conformation. If a protein is not correctly folded, it can lead to a condition known as the Unfolded Protein Response (UPR). However, whether UPR is activated depends on the nature of the protein. For instance, when proteins accumulate excessively, they may start to aggregate. It’s important to note that aggregation is not the same as forming condensates; rather, aggregation refers to multiple misfolded copies interacting with one another due to incorrect conformations, often caused by mutations. This altered structure can trigger 7 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 aggregation, which is considered a "gain of function" phenomenon. When a cell detects such aggregates or excess proteins that could be toxic, it can initiate the UPR. This response aims to restore normal function but does not exclude other mechanisms, such as the export and clearance of the misfolded proteins via the proteasome. If a protein is improperly folded, it may be transported out of the ER for degradation by the proteasome, which is distinct from lysosomal degradation. The proteasome is a protein complex rather than an organelle, as it is not surrounded by a membrane. It is a stable structure composed of multiple proteins, distinguishing it from aggregates or biomolecular condensates. For a protein to exit the endoplasmic reticulum (ER), it must be unfolded, even if it was initially folded incorrectly. This process requires energy, specifically in the form of ATP. Once the protein is properly unfolded, it can be directed to the proteasome for degradation. The proteasome recognizes polyubiquitinated proteins, which are marked for degradation, ensuring that misfolded or excess proteins are effectively cleared from the cell. The polyubiquitin chain we discussed is formed by the attachment of multiple ubiquitin molecules, each consisting of 76 amino acids. These ubiquitins are linked together in various configurations, depending on the specific regulatory functions they serve. In the context of protein degradation, a polyubiquitin chain is recognized by the proteasome, which facilitates the degradation of misfolded or excess proteins. As we mentioned yesterday, even though this polyubiquitin chain consists of multiple units, it is generally recognized by the proteasome as a single signal for degradation. However, it’s important to note that the activation of the Unfolded Protein Response (UPR) is not an automatic consequence of having a misfolded protein in the ER. While the presence of improperly folded proteins can trigger the UPR, this response does not necessarily activate every time a protein is not correctly folded. The response to misfolded proteins depends on several factors, including the nature of the protein, the extent of its accumulation in the endoplasmic reticulum (ER), and the specific cell type. It's important to consider that non-dividing cells, such as neurons and muscle cells, often exhibit stronger responses to protein misfolding because they cannot effectively manage toxic protein accumulation. In contrast, dividing cells may be less responsive to the Unfolded Protein Response (UPR) because they can dilute the problem through cell division. The context of the tissue and the specific physiological conditions play a significant role in how cells respond to stress. This leads us to the concepts of adaptive and maladaptive responses. Adaptive responses are temporary and help resolve stress; they represent a transient mechanism that allows the cell to cope with challenges effectively. Maladaptive responses, on the other hand, are chronic. In this case, the cell may be continuously activated in an attempt to manage the stress, but this prolonged activation does not lead to beneficial outcomes and can ultimately be detrimental. A maladaptive response occurs when chronic stress becomes problematic for the cell. While one might expect that every instance of stress or the presence of misfolded proteins could be effectively managed, this is not the case. The outcome depends on various factors, including the type of cell, the specific protein involved, and the conditions within the endoplasmic reticulum (ER). Therefore, it’s crucial to understand that not all stress responses lead to successful outcomes, and the context significantly influences how a cell copes with these challenges. (Alberts, strano) 8 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 In the image above, you can see the three different branches of the Unfolded Protein Response (UPR). I want to highlight these mechanisms in more detail because they represent a promising area of research aimed at enhancing the UPR to alleviate cellular stress. However, there are several limitations to consider. The three branches of the UPR include: 1. IRE1: This pathway ultimately leads to the production of chaperones that assist in protein folding within the ER. 2. PERK: This branch reduces overall protein translation to help manage stress. 3. ATF6: This pathway activates various other responses within the ER. Determining which of these branches is activated at any given time, and how they balance each other, is quite complex. Each cell may respond differently to stress by activating these interconnected pathways. For instance, if a drug is introduced that targets one of these pathways to enhance the UPR, the outcome is not guaranteed to be beneficial. In biology, responses are inherently regulated by various factors, including timing, spatial context, and the level of activation or down-regulation. When a drug is used to interfere with a dysfunctional process—such as one altered by mutations or disease—it can disturb the existing equilibrium. This alteration complicates predictions about the overall outcome, making it challenging to understand how such interventions will affect cellular health. This is why, during preclinical or clinical trials, it is essential to assess both safety and efficacy. Any intervention can alter various cellular responses within tissues, leading to complex outcomes. For example, let’s revisit the first branch of the Unfolded Protein Response (UPR), which promotes the production of chaperones that assist in protein folding. Then, we have the second branch, involving PERK, which is a kinase that phosphorylates eukaryotic elongation factor 2 (eEF2). Does anyone recall what eEF2 does? It plays a crucial role in the initiation of translation. When eEF2 is phosphorylated, it effectively downregulates translation. This happens because the phosphorylated form of eEF2 cannot efficiently bind to the Kozak sequence of mRNA, which is essential for the proper initiation of translation. As a result, the overall translation process is attenuated, limiting the production of new proteins in the cell. What happens is that translation slows down, but it doesn’t stop completely. Why not? Well, if translation were fully shut down, the cell wouldn’t produce anything at all, and that’s not what we want. Instead, translation is selectively attenuated. It’s slowed down, but it’s not off. This allows for the production of specific proteins, like chaperones, which help the cell manage stress, folding misfolded proteins, and preventing aggregation. These proteins are critical for dealing with the stress situation in the cell. For instance, In neurodegenerative diseases such as ALS, neurons face stress from misfolded or aggregated proteins, triggering the unfolded protein response (UPR) to manage these proteins. The UPR temporarily reduces protein synthesis, preventing the accumulation of misfolded proteins, which could otherwise overwhelm cellular machinery. eIF2-alpha phosphorylation is a key mechanism that reduces translation during stress, limiting the production of new proteins. This inhibition is selective; essential proteins like chaperones, which help with protein folding, can still be synthesized. GADD34 reverses eIF2-alpha phosphorylation, resuming normal translation. An inhibitor of GADD34 could keep eIF2-alpha phosphorylated longer, extending the reduction in protein synthesis to help the cell manage stress. Inhibiting GADD34 could, theoretically, slow down protein synthesis, giving neurons more time to process misfolded proteins. However, blocking GADD34 could have unpredictable effects in other tissues, as it impacts basic cellular functions, creating a risk for side effects. Peroxisomes Peroxisomes are fascinating and essential organelles in cell biology, yet they don’t always get the attention they deserve. They play crucial roles in cellular functions, and we can appreciate their importance by looking at the human disorders that arise when peroxisomes don’t function properly. So, what are peroxisomes? These are small, membrane-bound organelles found in almost all eukaryotic cells. Peroxisomes are especially vital for metabolizing fatty acids and detoxifying harmful substances. They carry out reactions that generate hydrogen peroxide—a potentially dangerous byproduct—and then use an enzyme called catalase to convert it into water and oxygen, which is harmless to the cell. When these reactions are disrupted, it can lead to a range of metabolic disorders. 9 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 When studying peroxisomes under a microscope, we often use advanced techniques like electron microscopy, which offers incredibly high resolution, essential for observing such small structures. For example, a scale bar in the image might indicate 200 nanometers, giving you a sense of size: molecules and organelles are visible at this level. Understanding microscopy resolution is important because it helps us know what size structures we're viewing. If the bar shows micrometers, you're looking at larger organelles grouped together. But if the bar shows nanometers, it means you're looking at much smaller structures or even individual molecules within an organelle. Techniques like electron microscopy are among the few that can provide the nanometer-level detail needed to visualize peroxisomes and similar small organelles. In summary, peroxisomes are tiny but powerful organelles essential for breaking down fatty acids and detoxifying cells, making them indispensable for cellular health. Notice that peroxisomes are highly electron-dense. Why? Because they’re packed with a lot of material. What kind of material? Mainly enzymes and lipids. So, let’s focus on their two main functions. First, detoxification. This function is somewhat similar to the smooth endoplasmic reticulum (ER), which also helps detoxify. In peroxisomes, detoxification involves oxidizing various molecules to form hydrogen peroxide (H₂O₂), a reactive oxygen species. However, since H₂O₂ is chemically reactive and can be harmful, peroxisomes contain an enzyme, catalase, to convert it into water (H₂O), rendering it safe for the cell. Anyways, peroxisomes are not just detoxifying organelles; they’re also vital for biosynthesis. Specifically, they’re involved in the breakdown of fatty acids through a process called beta-oxidation. In this process, long fatty acid chains are imported into peroxisomes and then chemically broken down. This involves breaking covalent bonds in the fatty acids, which produces shorter-chain fatty acids, acetyl-CoA, and intermediate molecules like plasmalogens—essential lipids for cell membranes, particularly for the myelin that surrounds neurons in the central and peripheral nervous systems. The acetyl-CoA generated can enter other metabolic pathways, such as glycolysis or the Krebs cycle, to support cellular energy production. In this sense, peroxisomes are not only detoxifying but also metabolic organelles— integral for both clearing harmful substances and contributing to cellular energy and biosynthesis. How are peroxisomes formed? Their formation is unique compared to other organelles. Organelles like the ER (endoplasmic reticulum) and mitochondria typically expand or replicate based on pre-existing structures: for example, new mitochondria arise from the division of existing mitochondria, and the ER expands from existing ER networks. In contrast, peroxisomes and lysosomes can form de novo, meaning they can be generated from scratch through the fusion of smaller vesicles. This allows the cell to produce new peroxisomes as needed without relying solely on the division of existing peroxisomes. Similarly, lysosomes can also form via this vesicle fusion process. This independence in formation provides flexibility, allowing the cell to adapt to varying metabolic and detoxification demands. Peroxisomes are organelles formed through the fusion of various vesicles, primarily derived from the endoplasmic reticulum (ER). This fusion process is flexible, as the peroxisome receives additional vesicles containing proteins and enzymes essential for its function. Peroxisomes can also undergo division, like mitochondria, to form new peroxisomes when the cell needs more of them. The red and green markers in diagrams of peroxisomes represent transport proteins on their membrane. These peroxins (PEX proteins), such as PEX1, PEX2, PEX7, and PEX9, are essential for transporting proteins and fatty acids from the cytosol into the peroxisome. Proteins imported this way must be unfolded before entry, while proteins already embedded in the peroxisome membrane come from ER-derived vesicles. The fusion of these small vesicles builds up the peroxisome. Peroxins are critical for peroxisome function. When peroxins are mutated, the peroxisome cannot import essential fatty acids or enzymes properly. This malfunction leads to the accumulation of long-chain fatty acids 10 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 in the cell, which are toxic when not broken down, and prevents the production of plasmalogens—key components of cell membranes, especially in the nervous system. 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 rely on protein translocation not just for structure but to remain fully functional. Why do mitochondria need to import so many materials? Although mitochondria contain their own circular DNA and multiple copies of it within each organelle, this DNA only encodes a limited set of proteins, primarily tRNAs and some regulatory molecules. This small mitochondrial genome has relatively few genes, encoding only a small portion of the proteins mitochondria need. It’s important to understand that a gene isn’t limited to encoding mRNA; it can also correspond to ribosomal RNA, transfer RNA, microRNA, and other RNA types. However, the mitochondrial genome has limited capacity to produce these components internally. For example, mitochondria depend heavily on proteins needed for oxidative phosphorylation, an essential energy-generating process that takes place in the cristae (the folds within the mitochondria). Many of these proteins must be imported from the cytosol, where they are synthesized. This process of importing necessary components into mitochondria is tightly regulated to ensure that each protein reaches its correct destination within the mitochondria, allowing them to maintain efficient energy production and other vital functions. Remember, mitochondria have a double membrane structure: an outer membrane, an inner membrane (which forms the cristae where oxidative phosphorylation takes place), and the matrix, the innermost compartment filled with mitochondrial material. These three areas serve different functions, and proteins must be precisely directed to each specific location—the outer membrane, the inner membrane (including the cristae), or the matrix. How are proteins selectively directed to each of these destinations? Mitochondria use specialized translocator complexes, which transport proteins to their correct location within the organelle. This transport process always requires energy. To pass through these membranes, the incoming proteins are unfolded by necessity, as fully folded proteins cannot pass directly into any organelle without being encapsulated in a vesicle. The TOM complex on the outer membrane is the first to receive these unfolded proteins from the cytosol, recognizing them through specific receptors, adapters, and interactors. Although complex, this system of transport and recognition ensures high regulation, directing proteins exactly where they are needed to maintain mitochondrial function. So each component in the mitochondrial transport system works together—receptors, interactors, and more— in a way that carefully regulates protein import. Just because proteins are near the mitochondria doesn’t mean 11 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 they’ll automatically enter. Although the space around mitochondria is densely packed with molecules moving and interacting, only certain proteins are imported, and this selection depends on specific interactions. To control what gets imported, proteins destined for the mitochondria have a signal sequence—a unique amino acid stretch that identifies them as ideal candidates for import. This sequence is recognized by a receptor on the mitochondria, which initiates the import process. The receptor then interacts with a translocator complex to guide the protein inside. This system keeps the process selective and well-regulated. The main translocator complexes have distinctive names: TOM (Translocase of the Outer Membrane) operates on the outer membrane, while others like TIM and OXA handle inner membrane and matrix transport. For instance, the TOM complex initially recognizes the protein’s signal sequence through a receptor and hands it off to further complexes, depending on its final destination within the mitochondria. This process ensures that only the correct proteins reach the right mitochondrial compartments, all while maintaining efficient regulation. If a protein needs to be delivered to the inner membrane, such as the cristae, it is transferred from the TOM complex to the TIM complex. The TIM complex then directs the protein to its appropriate location using either the TIM22 or OXA complex. This indicates that these complexes must interact with each other to ensure that the import into the inner membrane occurs specifically at the contact points between the two membranes, rather than randomly. Now, let's talk about energy requirements. Any cellular process that involves creating covalent bonds or phosphorylation demands energy. In this context, the transport process is classified as active transport, meaning it requires energy. What is the source of this energy? Typically, it comes from ATP. The hydrolysis of ATP produces ADP and releases inorganic phosphate. Additionally, transport can utilize the membrane potential. Speaking of membrane potential, it's essential to understand that mitochondria possess a membrane potential. In fact, all organelles have a membrane potential. (Can anyone recall which other membranes in the cell also have a membrane potential? The plasma membrane.) 12 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 So, the differences in ion distribution between the inside and outside of the cell create a membrane potential, which is a key example of asymmetry. The inside of the cell is negatively charged, primarily due to the presence of negatively charged ions and phosphatidylserine located in the inner leaflet of the plasma membrane. The plasma membrane has a membrane potential, but it's important to note that while all organelles exhibit some degree of asymmetry, not all have a significant membrane potential. In contrast, the inner membrane of the mitochondria has a much higher membrane potential, typically around 100 to 130 millivolts. This higher potential arises from processes occurring in the intermembrane space during oxidative phosphorylation. Protons are pumped into this space, creating a charge differential. This electrochemical gradient is crucial, as it provides the energy necessary for various transport processes. The membrane potential serves as an energy source, enabling transporters to import essential substances into the mitochondria. Additionally, oxidative and reductive reactions are coupled with these transport processes, further facilitating energy transfer for transport functions. Gated regulated transport: the nucleus We're discussing gated regulated transport, a mechanism essential for maintaining the function of both the nucleus and cytosol by enabling material exchange. Now, what types of materials are transported across the nuclear envelope? Let’s start with histones, which direction? From the cytosol to the nucleus. And RNA? Which direction? From the nucleus to the cytosol. In addition to histones and RNA, other important materials must cross into or out of the nucleus: numerous proteins like polymerases, essential for DNA replication and transcription, along with splicing factors, enzymes, and various metabolites involved in nucleic acid metabolism. There’s something else critical here— what about nucleotides? Yes, exactly. Nucleotides serve as a source of energy and building blocks. Now, let’s talk about something larger in scale: biomolecular condensates. What's an example of a biomolecular condensate in the nucleus? The nucleolus. This is where ribosomal RNA (rRNA) and ribosomal proteins are assembled into ribosomes. These ribosomes, once assembled, need to exit the nucleus and enter the cytosol to function. This transport of ribosomes requires passing a large mass through nuclear pores, meaning these pores must be substantial enough to accommodate complex assemblies of rRNA and proteins. So, nuclear pores play a crucial role as they connect the inner and outer nuclear membranes, allowing these large molecular assemblies to move in and out of the nucleus. Nuclear pores are formed by proteins called nucleoporins. These nucleoporins are diverse but assemble in large numbers, creating a structure resembling a complex gate with a mesh-like quality. How does this structure function? Nucleoporins contain unstructured regions, which give them a flexible, fibrillar appearance. This lack of fixed shape allows them to form a mesh that facilitates transport. These regions are rich in specific amino acids, especially phenylalanine and glycine, which help nucleoporins accommodate various cargoes, such as proteins that need to be imported from the cytosol into the nucleus. Electron microscopy reveals the arrangement of nucleoporins, showing a rosette-like structure when viewed from above, with pores at the center and various protruding fibrils. How does transport occur through this pore? As we discussed, each cargo protein has a signal that is recognized by specific nuclear import receptors, which guide them through the nucleoporin mesh. Multiple cargo types can be recognized by different receptors, each navigating through this structure in a regulated process. Is this transport energy-dependent? Absolutely. In this case, the energy comes not from ATP, but from the action of a small GTPase, which provides the necessary drive for regulated, directional movement through the nuclear pore 13 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 These small GTPases play crucial roles in cellular regulation, particularly in trafficking. Take, for example, the GTPases Rho, CDC42, and RAC. These proteins toggle between an active GTP-bound state and an inactive GDP-bound state. This shift happens because a GTPase can hydrolyze bound GTP, converting it into GDP. However, to re-activate the GTPase, GDP must be exchanged for GTP, a process enabled by GEFs (Guanine nucleotide Exchange Factors). One example of this process involves the GTPase Ran. Inside the nucleus, chromatin scaffolds support a protein called Ran GEF, which catalyzes the exchange of GDP for GTP on Ran. Ran enters the nucleus in its inactive GDP-bound form, but upon contact with Ran GEF, it releases GDP and binds GTP, becoming active. This activated Ran GTP interacts with cargo molecules, enabling them to travel through the nuclear pore complex. Once in the cytosol, Ran GTP undergoes hydrolysis, releasing one phosphate to convert back to Ran GDP. This cycle keeps Ran GDP concentrated in the cytosol and Ran GTP in the nucleus. This gradient is essential, as it helps regulate the directional movement of cargo into and out of the nucleus, maintaining the balance of transport within the cell. (Alberts) 14 Molecular cell biology-BolinoLezione n°2 11 Ottobre 2024 We discussed the need to transport various components such as mRNA, ribosomes, transcription factors, polymerases, and other essential proteins involved in DNA replication, transcription, translation, RNA maturation, and more. But these nuclear pores, or 'gates,' do more than just facilitate this bidirectional transport of materials between the nucleus and cytosol. They also play a crucial role in regulating gene transcription and nucleotide function within the nucleus. A good example is calcineurin. Unlike calnexin, calcineurin is a phosphatase activated by calcium. Once activated, calcineurin dephosphorylates specific substrates, including a transcription factor called NFAT (or NFAT-C4, depending on the cell type). This transcription factor is highly influential, acting as a master regulator of various gene transcription programs. When phosphorylated, NFAT-C4 remains inactive in the cytosol. However, upon activation by calcineurin, the phosphate groups are removed, activating NFAT-C4 and allowing it to enter the nucleus where it can initiate downstream gene transcription. This selective gating allows cells to compartmentalize certain proteins, keeping them inactive in the cytosol until needed in the nucleus. Thus, the pores not only control the flow of necessary components but also regulate the timing and location of protein activity within the nucleus. This ensures that transcription factors and other regulatory proteins only become active when and where they are required, adding an additional layer of control to cellular function. Next time we will enter, finally, how the compartments act. 15