BM MT 2 Micrographs Spring 2024 PDF

MT 2 Micrographs & Diagrams  Pinocytosis. a. Micropinocytosis involves the b. This electron micrograph shows c. Macropinocytosis – involves the dynamic formation of small vesicles numerous smooth-surfaced...

MT 2 Micrographs & Diagrams  Pinocytosis. a. Micropinocytosis involves the b. This electron micrograph shows c. Macropinocytosis – involves the dynamic formation of small vesicles numerous smooth-surfaced rearrangement of the plasma membrane at the cell surface. First, substances pinocytotic vesicles(arrows) within and underlying actin cytoskeleton to form to be pinocytosed the cytoplasm of endothelial cells of surface membrane ruffles that entrap invaginated, and a blood vessel. Also membrane large volumes of extracellular fluid. The dynamin (a GTPase) pinches off ruffles are visible on this image. large vesicles (macrospinosomes) the vesicle from the membrane They are essential in formation of enter the cell cytoplasm, to become a pinocytotic vesicle large macrospinosomes. X55 000. undergo maturation, and within the cell. ✔ either fuse with early lysosomes or Pinocytosis of certain substances ✔ return to the plasma membrane for may be associated with caveolin. recycling.  Phagocytosis [Gr., cell eating] Is the ingestion of large particles such as ✔ cell debris, ✔ bacteria, and ✔ other foreign materials. Plasma membrane sends out PSEUDOPODIA to engulf phagocytosed particles into large vesicles (larger than approximately 250 nm in diameter) called phagosomes. Is performed mainly by a specialized group of cells belonging to the Mononuclear Phagocyte System (MPS). Is a receptor-mediated process ✔ Antibodies coat the surface of an invading microorganism or cell ✔ Receptors on the surface of the phagocyte cells recognize the Fc portion of the antibodies – non – antigen-binding domains (Fc fragments) of antibodies (a). ✔ This interaction triggers rearrangement of actin cytoskeleton. Depolymerizations and repolymerizations of actin filaments produce temporary projections of the plasma membrane called pseudopodia. They surround phagocytosed particle and lead to the formation of phagosomes. By targeted delivery of lysosomal enzymes, a phagosome matures into a lysosome Recognition of PAMPs leads to activation that digests its phagocytosed content. of nuclear factor kappa B (NF-B) transcription factor, which regulates genes Is also triggered by recognition of that control cell responses in phagocytosis. ▪ PAMPs - Pathogen Associated Molecular Patterns - expressed on pathogen surfaces by ▪ Toll-like receptors - expressed on macrophages.  Nonbiologic materials Biologic debris from such as o inflammation o inhaled carbon particles o wound healing o inorganic dusts o dead cells o asbestos fibers are sequestered by cells of the Mononuclear Phagocyte System (MPS) without involvement of Fc receptors (b). Initial pseudopodial extensions of plasma membrane are based on the actin cytoskeleton rearrangement - depolymerization and repolymerization of the actin filaments. Phagocytosis is referred to as clathrin-independent but actin-dependent endocytosis!!! a. Receptor-mediated endocytosis. A fully formed coated pit pinches off from the plasma membrane by the protein complex dynamin. Peripheral membrane protein - Dynamin - Forms contracting loops around the developing neck of the pit; - This neck cause the region to pinch off as a coated vesicle. Selected cargo proteins and their receptors are pulled from the extracellular space into the lumen of a forming coated vesicle. After budding and internalization of the vesicle, the coat proteins are removed and recycled for further use. The uncoated vesicle travels to its destination to fuse with a cytoplasmic organelle. The type of vesicle formed as a result of receptor-mediated endocytosis is referred to as a coated vesicle, and the process itself is known as clathrin- dependent endocytosis. Clathrin-coated vesicles are also involved in the movement of the cargo material ✔ from the plasma membrane to early endosomes ✔ from the Golgi apparatus to the early and late endosomes. Exocytosis Intracellular traffic of vesicles is achieved by the presence of specific proteins on their surface COATOMERS such as ▪ COP-I ▪ COP-II ▪ Newly synthesized proteins after their initial posttranslational modification, are delivered in COP-II – Coated Vesicles to the Golgi apparatus ▪ Retrograde transport is present between Golgi cisternae and is mediated by the COP-I – Coated Vesicle. The molecules that travel this route are often chemically modified (e.g., glycosylated, sulfated) as they pass through different cellular compartments. The membrane that is added to the plasma membrane by exocytosis is recovered into the cytoplasmic compartment by an endocytotic process. 2 general pathways of exocytosis: 1. The constitutive pathway ▪ Substances designated for 2. The regulated pathway export are continuously delivered They concentrate secretory in transport vesicles to the plasma proteins and transiently store membrane. them in secretory vesicles within ▪ Proteins that leave the cell by the cytoplasm. this process Regulatory hormonal or - are secreted immediately neural stimulus after their synthesis and ▪ causes a transient influx of - exit from the Golgi apparatus Ca2+ into the cytoplasm ▪ Ca2+ stimulates secretory vesicles to fuse with the plasma membrane and discharge their contents. Secretory vesicles containing inactive precursor (zymogen) were called zymogen granules.  Steps in formation, targeting, docking, and fusion of transport vesicles with the target membrane. 8. Formation of the docking complex between ▪ Rab-GTPase and its protein in the target membrane ▪ v-SNAREs on the immobilized vesicle interact with t-SNAREs on the target membrane to form the cis-SNARE complex. SNAREs ▪ Are transmembrane proteins grouped according to their location within the vesicle (v-SNARE) or target membrane (t-SNARE). ▪ Guarantee the specificity of interaction between a particular vesicle and its target membrane ▪ Promote membrane fusion that follows immediately after the cis-SNARE complexes are formed. 9. Fusion of the vesicle to the targe 1. Lipid raft with cargo receptors ready to interact 5. Disassembly of clathrin coat and expression of membrane. with cargo protein. Note the presence of the specific Rab-GTPase activity – target recognition protein. 10. Discharge of the cargo protein targeting protein v-SNARE ("SNAp REceptor" – a large 6. Tethering of the vesicle to the target membrane by into the early endosomal protein family consisting of at least 24 members in the interaction between Rab-GTPase and tethering compartment and disassembly of yeasts, more than 60 members in mammalian cells, proteins. the cis complex by the interaction and some numbers in plants). 7. Beginning of the docking process (recruitment of of the NSF/α-SNAP protein 2. Initial step in vesicle formation: The binding of the tethering proteins): complex. adaptin complex and clathrin forms a coated pit. ▪ Recognition of the vesicle by initial 11. Recycling of v-SNAREs in the 3. Formation (budding) of fully assembled coated interaction/tethering transport vesicles for use in vesicle. ▪ Recruitment of the necessary number of another round of vesicle targeting 4. Coated vesicle travels to its destination. tethering proteins to dock the incoming vesicle. and fusion.  How the Endosomes become the lysosomes Some endosomes also communicate with the vesicular transport system of the rER. This pathway provides constant delivery of newly synthesized lysosomal enzymes, or hydrolases. Hydrolase synthesis in the rER as an enzymatically inactive precursor called a prohydrolase. Folding of this heavily glycosylated protein. Creation of a signal patch / recognition signal ▪ due to the approach of certain amino acids in close proximity & ▪ its exposure on the surface of the protein molecule. Modification of the signal patch on a protein destined for a lysosome. ▪ Attachment of mannose- 6-phosphate (M-6-P) to the prohydrolase surface (M-6-P acts as a target for proteins possessing an M-6-P receptor). ▪ M-6-P receptors are present in Later these vesicles are transported to the early or late ✔ early and late endosomes, endosomes ✔ lysosomes, and ✔ the Golgi apparatus. The acidic environment of late endosomes causes the release of prohydrolases from the M-6-P receptors. Sorting and packaging of the lysosomal enzymes into vesicles in the TGN of the Prohydrolases are next activated by cleavage and by removing Golgi apparatus. phosphate groups from the mannose residues Early endosomes Late endosomes 1. Can be found in the more 1. Are often positioned near the peripheral cytoplasm Golgi apparatus and the nucleus. 2. Have a tubulovesicular structure: 2. Have a more complex structure The lumen is subdivided into and often exhibit onion-like cisternae that are separated by internal membranes. invagination of its membrane. 3. Exhibits only a slightly more 3. Their pH is more acidic, acidic environment (pH 6.2 to 6.5) averaging 5.5. than the cytoplasm of the cell. 4. The major function of early 4. Late endosomes –prelysosomes- endosomes is to sort and recycle mature into lysosomes, may fuse proteins internalized by with each other or with mature endocytotic pathways. lysosomes. 5. Their morphologic shape and 5. Substances transported to late geometry of the tubules and endosomes are degraded in vesicles create an environment in lysosomes. which localized changes mechanism includes dissociation of ligands from their receptor protein; 6. The narrow diameter of the tubules and vesicles may also aid in the sorting of large molecules, Diagram shows the fate of protein (red circles) endocytosed from the cell surface and destined for which can be mechanically lysosomal destruction. Proteins are first found in endocytotic (coated) vesicles that deliver them to prevented from entering specific early endosomes, which are located in the peripheral part of cytoplasm. Because of the sorting sorting compartments. capability of the early endosomes, receptors are usually recycled to the plasma membrane, and 7. After sorting, most of the protein endocytosed proteins are transported via multivesicular bodies (MVB) to late endosomes positioned is rapidly recycled, and the excess near the Golgi apparatus and the nucleus. The proteins transported to late endosomes eventually will membrane is returned to the be degraded in lysosomes. Note the acidification scale (left) that illustrates changes of pH from early plasma membrane. endosomes to lysosomes. The acidification is accomplished by the active transport of protons into endosomal compartments. Fate of receptor and ligand in receptor-mediated endocytosis. This diagram shows four major pathways along which the fate of internalized ligand–receptor complexes is determined. a. The internalized ligand–receptor complex dissociates, the receptor is recycled to the cell surface, and the ligand is directed to late endosomes and eventually degraded within lysosomes. This processing pathway is used by the LDL–receptor complex, insulin–GLUT receptor complex, and a variety of peptide hormone–receptor complexes. LDL, low-density lipoprotein; MVB, multivesicularbodies. b. Both internalized receptor and ligand are recycled. Ligand–receptor complex dissociation does not occur, and the entire complex is recycled to the surface. An example is the iron–transferrin–transferrin receptor complex that uses this processing pathway. Once iron (Fe) is released inthe endosome, the transferrin–transferrin receptor complex returns to the cell surface, where transferrin is released. c. The internalized ligand–receptor complex dissociates in the early endosome. The free ligand and the receptor are directed to the late endosomal compartment for further degradation. This pathway is used by many growth factors (i.e., the EGF–receptor complex). d. The internalized ligand–receptor complex is transported through the cell. Dissociation does not occur, and the entire complex undergoes transcytosis and release at a diff erent site of the cell surface. This pathway is used during secretion of immunoglobulins (secretory IgA) into saliva. The antibody IgA–receptor complex is internalized at the basal surface of the secretory cells in the salivary gland and released at the apical surface. Lysosome biogenesis: Delivery of Lysosomal The lysosomal membrane possesses highly glycosylated specific membrane proteins that protect the membrane from digestion by lysosomal enzymes. Specific Membrane Proteins. These lysosomespecific proteins are synthesized in the rough endoplasmic reticulum, transported to the Golgi apparatus, and reach their destination by 2 pathways: 1. Blue arrows indicate the Constitutive Secretory Pathway in which ▪ certain lysosomal membrane proteins LIMPs (lysosomal integral membrane proteins) exit the Golgi apparatus and ▪ are delivered to the cell surface. ▪ From there they are endocytosed and, via the early and late endosomal compartments, finally reach lysosomes. 2. Green arrows indicate the Endosomal Golgi-Derived Coated Vesicle Secretory Pathway. ▪ Here, LIMPs, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles. ▪ These transport vesicles travel and fuse with late endosomes as a result of interaction between endosome-specific components of v-SNARE and t-SNARE docking proteins. Lysosome biogenesis - delivery of lysosomal enzymes This diagram shows The intracellular trafficking leading to the delivery of many soluble lysosomal enzymes to late endosomes and regulated and lysosomes involves the M-6-P signal and its receptor. constitutive Hydrolase synthesis in the rER as an enzymatically Pathways for delivery inactive precursor called a prohydrolase. of lysosomal specific Folding of this heavily glycosylated protein - enzime. membrane proteins Creation of a signal patch / recognition signal due to into early and late the approach of certain amino acids in close proximity and endosomes. its exposure on the surface of the enzime molecule. Modification of the signal patch on a protein destined for a lysosome. Attachment of mannose- 6-phosphate (M-6-P) to the prohydrolase surface (M-6-P acts as a target for proteins possessing an M-6-P receptor). M-6-P receptors are present in ✔ early and late endosomes, Release of Activation ✔ lysosomes, and prohydrolases from of prohydrolases by ✔ the Golgi apparatus. the M-6-P receptors cleavage and by Sorting and packaging of the lysosomal enzymes into due to the acidic removing phosphate vesicles in the TGN of the Golgi apparatus. environment of late groups from the Transport of these vesicles to the early or late endosomes. endosomes. mannose residues. Depending on the nature of the digested material, 3 different pathways deliver material for intracellular digestion in lysosomes. Phagocytosis - Extracellular large particles such as ▪ bacteria, ▪ cell debris, and ▪ other foreign materials are engulfed. ▪ A phagosome, formed as the material is internalized within the cytoplasm, subsequently receives hydrolytic enzymes to become a late endosome, which matures into a lysosome. Pinocytosis & Receptor - Mediated Endocytosis - Extracellular small particles such as ▪ extracellular proteins, ▪ plasma-membrane proteins, and ▪ ligand–receptor complexes are internalized. ▪ These particles follow the endocytotic pathway through early and late endosomal compartments and are finally degraded in lysosomes. Autophagy - Intracellular particles such as ▪ entire organelles, ▪ cytoplasmic proteins, and ▪ other cellular components ▪ They are isolated from the cytoplasmic matrix by sER membranes, transported to lysosomes, and degraded. Some cells (e.g., osteoclasts involved in bone resorption and neutrophils involved in acute inflammation) may release lysosomal enzymes directly into the extracellular space to digest components of the tracellular matrix.  3 Autophagic Pathways for Degradation of Cytoplasmic Constituents. 1. Macroautophagy / simply autophagy Is a nonspecific process in which ▪ A portion of the cytoplasm or an entire organelle is surrounded by an intracellular membrane of the endoplasmic reticulum, called isolation membrane to form a double-membraned autophagosome vacuole. ▪ After fusion with a lysosome, the inner membrane and the contents of the vacuole are degraded. ▪ This process is aided by proteins encoded by several Atg genes: - the complex containing Atg12–Atg5–Atg16L proteins attaches to a part of endoplasmic reticulum and localizes the isolation membrane. - Subsequently, Atg8 is recruited and bound to the membrane. - Together they change the shape of the isolation membrane, which bends to enclose and seal an organelle destined for digestion within the lumen of the autophagosome. - Once the autophagosome is completed, the Atg12–Atg5–Atg16L complex and Atg8 dissociate from this structure. - After targeted delivery of lysosomal enzymes, the autophagosome matures into a lysosome. - The isolation membrane disintegrates within the hydrolytic compartment of a lysosome. 2. Microautophagy ▪ is a nonspecific process ▪ small cytoplasmic soluble proteins are degraded in a slow,continuous process under normal physiologic conditions. ▪ They are internalized into the lysosomes by invagination of the lysosomal membrane. 3. Chaperone-mediated autophagy ▪ is the most selective process for degradation of specific cytoplasmic proteins. ▪ It requires assistance of proteins called chaperones. ▪ The chaperone protein— hsc73 ✔ binds to the protein and ✔ helps transport it into the lysosomal lumen, where it is finally degraded. ▪ This process is activated during nutrient deprivation. ▪ It requires: ✔ targeting signals on the degraded proteins and ✔ a specific receptor on the lysosomal membrane. ▪ Chaperone-mediated autophagy is responsible for the degradation of approximately 30% of cytoplasmic proteins in organs such as the liver and kidney. The Golgi apparatus and vesicular trafficking. M-6-P is added to those proteins destined to travel to late endosomes and lysosomes. Additionaly glycoproteins are phosphorylated and sulfated. The proteolytic cleavage of certain proteins is also initiated within the cisternae. Retrograde transport from CGN to rER, as well as retrograde transport between Golgi cisternae, is mediated by COP-I–coated vesicles. Once proteins have been modified within the CGN the transport vesicles bud off dilated ends of this compartment and proteins are transferred into medial-Golgi cisternae. The process continues; in the same fashion, proteins are translocated into the trans-Golgi cisternae and further into the trans-Golgi network (TGN). In the TGN they are sorted into different transport vesicles that deliver them to their final destinations.  Summary of events in protein trafficking from the TGN. The tubulovesicular array of the TGN serves as the sorting station for transporting vesicles that deliver proteins to the following destinations: (1) Apical plasma membrane (i.e., epithelial cells); (2) Apical region of the cell cytoplasm where proteins are stored in secretory vesicles (i.e., secretory cells); (3) Early or late endosomal compartment; (4) Lysosomes - selected proteins containing lysosomal signals, are targeted to lysosomes; (5) Lateral plasma membrane (i.e., epithelial cells); (6) Basal plasma membrane (i.e., epithelial cells); (7) Basal plasma membrane - proteins destined for apical, basal, and lateral surfaces of plasma membrane, which are delivered to the basal plasma membrane (i.e., in hepatocytes); (8) all proteins endocytosed and sorted in early endosomes; From early endosomes – (9) apical plasma membrane, (10) lateral plasma membrane and (11) basal plasma membrane. ❖ Note two targeting mechanisms of proteins to different surfaces of plasma membrane: ▪ In epithelial cells, proteins are directly targeted from the TGN into ▪ In hepatocytes, all proteins are secreted first to the basal cell the appropriate cell surface as shown in steps (1), (5), and (6). surface, and then they are distributed to the appropriate cell surface via the endosomal compartment as shown in steps (7) to (11). https://jtw441.wordpres s.com/2014/02/04/roug h-endoplasmic-reticulum- rer-smooth-endoplasmic- reticulum-ser-and-riboso mes/ The endoplasmic reticulum (ER) The cytosolic compartment contains ▪ is an interconnected network of membrane-bound channels ▪ soluble proteins within the cytoplasm, ▪ cytoskeletal components ▪ Is part of the cytomembrane system and distinct from the ▪ organelles. plasma membrane. 2 types of endoplasmic reticulum: ▪ consists of cisternae (flat sacs), tubules and vesicles, 1. The rough endoplasmic reticulum (RER). ▪ divides the cytoplasm into two compartments: 2. The smooth endoplasmic reticulum (SER) 1. The luminal or endoplasmic compartment. Ergastoplasm - portion of cytoplasm containing RNA, stained 2. The cytoplasmic or cytosolic compartment. with the basic dye and recognized under the light microscopes. Groups of ribosomes form short spiral arrays called polyribosomes or polysomes in which many ribosomes are attached to a thread of messenger RNA (mRNA). This image shows a small section of the rER adjacent to the nucleus sectioned in two planes. The reticulum has turned within the section. ▪ In the upper right and left, the membranes of the reticulum have been cut at a right angle to their surface. ▪ In the center, the reticulum has twisted and is shown as in an aerial view (from above the membrane). ▪ The large spiral cytoplasmic assemblies (arrows) are chains of ribosomes that form polyribosomes that are actively engaged in translation of the mRNA molecule. 38,000. Polyribosomes (red arrow) located on a tangentially (a straight line or plane that touches a curve or curved surface at a point, but if extended does not cross it at that point) sectioned cistern of the granular endoplasmic reticulum from thalamocortical relay neuron. mRNA filament visible at blue arrow. Scale = 300 nm. (Rat, thalamic ventrobasal nucleus). http://synapses.clm.utexas.edu/atlas  A residual body ▪ a small vacuolar remnant ▪ contains indigestible material retained after release of nutrients into the cytosol through the lysosomal membrane. ▪ In some long-lived cells (eg,neurons, heart muscle), can accumulate over time granules of lipofuscin. Heterogeneity in the content of lysosomes near the plasma membrane between two hepatocytes. Some of the lysosomes contain residues resulting from partial digestion of lipids and some of these are more electron dense than others. Also in the field are mitochondria, rough endoplasmic reticulum, and the cell membrane. (George E. Palade; original is in the Palade Collection, University of California, San Diego). Electron micrograph of the Golgi apparatus. This electron micrograph shows the extensive Golgi apparatus in an islet cell of the pancreas. The flattened membrane sacs of the Golgi apparatus are arranged in layers. The CGN is represented by the flattened vesicles on the outer convex surface, whereas the flattened vesicles of the inner convex region constitute the TGN. Budding off the TGN are several vesicles (1). These vesicles are released (2) and eventually become secretory vesicles (3). x55,000. The cytoplasm of a liver cell. X27 000 Lysosomes Ly1 vary greatly in size and appearance but are Late endosomes or multivesicular bodies MB are also seen in this recognised as membrane-bound organelles containing an micrograph. Note the size of lysosomes relative to mitochondria M amorphous granular material. Multivesicular bodies (MBs) - A specialised subset of endosomes Phagolysosomes or secondary lysosomes Ly2 are even more that contain membrane-bound intraluminal vesicles. variable in appearance but are recognisable by their diverse These vesicles form by budding into the lumen of the MB. particulate content, some of which is extremely electron-dense. The content of MBs can be degraded, via fusion with lysosomes, or The distinction between residual bodies and secondary released into the extracellular space as the exosomes, via fusion lysosomes is often difficult. with the plasma membrane. Electron micrograph of nutrient-starved mouse embryonic fibroblast. This electron micrograph shows several autophagosomes 2 autophagosomes at the upper left contain portions of RER more (AP). Note that AP are double membraned structures containing electron dense than the neighboring normal RER. undigested intracellular organelles, such as mitochondria or One near the center contains what may be mitochondrial fragments of the endoplasmic reticulum (arrowheads). After the membranes plus RER. autophagosome fuses with a lysosome, it forms an autolysosome Also shown is a vesicle with features of a residual body (RB). (AL) that degrades the enclosed materials including the inner X30,000. autophagosomal membrane. M, mitochondria, rER, rough endoplasmic reticulum. X 26 300 (Pawlina, 2020). SER in a steroid-producing Leydig cell in the testis Electron micrograph of the sER. This image shows numerous profiles of sER in an interstitial (Leydig) cell of the testis, a cell that produces steroid hormones. The sER seen here is a complexm system of anastomosing tubules. The small, dense objects are glycogen particles. x60,000. The End.