Lecture 7 - Plasma Membrane Structure and Function (1) PDF
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This document is a lecture on plasma membrane structure and function, focusing on secretion pathways, lysosomes, and autophagy. It discusses various mechanisms and pathways within cells. The text includes details of the processes involved and examples.
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0/17/2024 1 ECTURE 7 - PLASMA MEMBRANE STRUCTURE AND FUNCTION L S ecretion at the plasma membrane allows for the transport of products made in the secretory pathway to the cell exterior ○ It al...
0/17/2024 1 ECTURE 7 - PLASMA MEMBRANE STRUCTURE AND FUNCTION L S ecretion at the plasma membrane allows for the transport of products made in the secretory pathway to the cell exterior ○ It also allows for the insertion of membrane proteins S ecretion pathways→ The default pathway of secretory vesicles;does not require any form of asignal ○ Pathways that direct these vesicles to other locations require signals L ysosomes→Dynamico rganelles that consist of hydrolytic enzymes (hydrolases) to break down waste/molecules ○ Prefer acidic conditions (pH of 4.5 to 5) ○ C onsists of different enzymes for the different components it can break down E.g. Proteases ⇔ Amino acids, Nucleases ⇔ nucleotides, etc ○ H as proton pumps which gathers protons into its lumen to achieve that acidic environment H-pump is driven by ATP hydrolysis Late endosome→ The stage prior to its maturation into a lysosome Has proton pumps that generate acidic conditions, but the pH is not low enough ○ T hese organelles are dynamic because lysosomes do not have a fixed appearance They mature overtime, altering size and shape ○ V esicles from the Golgi can fuse with lysosomes, enabling the vesicles’ components to further break down These vesicles now carry lysosomal enzymes There are four major pathways that are used to deliver materials to lysosomes ○ One internal and three external pathways ○ A utophagy→ The internal delivery pathway where cells dispose of obsolete components (e.g. macromolecules that are not functioning to their best potential) These defective materials areflagged(often by ubiquitination), enabling the formation of a autophagosome Autophagosome→ A combination of vesicles that recognise the flagged signal and bind together to form a double-layered membrane around the obsolete components This temporary vesicle fuses with a lysosome to initiate digestion This process is also known asselective autophagy N onselective autophagy→ Cell cultures are starved resulting in autophagosomes to consume large amounts of cytosol to help the cell sustain itself Acid hydrolases are made, N-glycosylated and folded in the ER ○ Next, they move into COP2 vesicles and are transported to the Golgi ○ M annose phosphorylation (M6P)→ Certain mannose sugars of the N-linked glycans are phosphorylated in the Cis-Golgi which signal the acid hydrolases to travel to lysosomes ○ A group of amino acids known as asignal patchverifies the signal on the target protein and determines whether it can undergo mannose phosphorylation (receive the M6P signal) ○ M 6P proteins bind toMPRs(mano-6-phosphate receptors) in the trans Golgi network The MPRs attached to acid hydrolases, then enter clathrin vesicles, which fuse with a late endosome to eventually evolve into a lysosome MRPs are recycle back to the TGN Vacuoles→ Sites of intracellular digestion in plants ○ Due to its ill taste, vacuoles help ward off predation ○ T hevacuolar targeting sequenceis a section of a nascent protein recognized bysorting proteinsat TGN These sequences are then carried to the vacuole where the v-targeting sequence is cleaved off and the protein matures This is unlike M6P as once the M6P signal reaches the lysosome, it permanently remains there There are different types of secretion: ○ Constitutive secretion→ Thecontinuouscreation and secretion of proteins in anunregulatedmanner Most plasma membrane and extracellular components move using this type secretion ○ R egulated secretion→ To be secreted proteins are packed into vesicles (secretory granules) and are sent to the extracellular matrix once asignalis recognized These signals can be a stimulus (e.g. variations in Ca ion concentration) M ost neurotransmitters, hormones and digestive enzymes use this form of secretion Cells are concentrated into vesicles using the following behaviours: ○ Similar products cluster together in a vesicle ○ C lathrin vesicles pinch off immature secretory vesicles which undergo retrograde movement to the TGN to return the components that are not supposed be in secretory vesicles Endocytic vesiclesare made at the plasma membrane ○ The fate of the substances transported from the extracellular matrix is determined bysorting at the early endosome If certain components remain in the early endosome, the endosome will merge with a pre-lysosome, evolve into alysosomeand degrade its contents ○ R ecycling endosome→ An endosome which transports the remnants within an early endosome back to the cell’s exterior P inocytosis→Cell drinking; Large amounts of substances around the plasma membrane is consumed by a cell ○ Pinocytic vesicles can and cannot be clathrin coated ○ Caveolaeare flask-like pinocytosis vesicles that are not coated by clathrin, They are coated by caveolin I and caveolin II ○ M acropinocytosis→ An endocytic pathway involving actin or cytoskeletal polymerization at the plasma membrane that are independent of clathrin-coated vesicles A signal activates the rearrangement of the actin cytoskeleton to form large ruffles or cell protrusions T he protrusions curve in towards the plasma membrane while capturing nearby cargo O nce the ruffle merges with the membrane, a vesicle (macropinosome) forms, allowing the collected cargo to be transported into the cell R eceptor mediated endocytosis→ A process that controls how cholesterol is taken up by cells (for example) ○ Cholesterol is packed and transported aslow-density lipoproteins (LDL) ○ W hen cholesterol is required,LDL-receptorsare increased on the plasma membrane The LDL interacts with its receptors to form clathrin-coated vesicles which uncoat and transport the contents to an early endosome D ue to the difference in pH between an endosome and cytosol, the costly LDL receptors are not degraded, but return to the plasma membrane for reuse T he lipoproteins in the early endosome are digested by hydrolases o nce the endosome evolves into a lysosomes The digestion results in the release of cholesterol into the cytosol ○ Just to clarify, this is an example of receptor-mediated endocytosis E ndocytic receptorsc an be recycledback to the plasma membrane,or can be degraded by lysosomes ○ Epidermal growth factor receptors is an example of a receptor that is tagged, transported to the lysosome and degraded P hagocytosis→ Endocytosis carried out by cells with specialized immune functions (e.g. macrophages) ○ Starts off with the formation or a large vesicles calledphagosomeswhich enclose the cargo and bring it into the cell The phagosome is large enough to consume bacteria ○ This endocytic vesicle then fuses with a lysosome to degrade its contents There are consequences when lysosomal enzymes do not reach lysosomes ○ Lysosomal storage diseases → The accumulation of waste in lysosomes causes them to swell and eventually burst, killing the cell E.g. I-cell diseasecauses severe skeletal and developmental abnormalities P atients have mutations in a gene that would normally enable M6P signals (mutant cis golgi enzyme) Thus, they do not have the enzyme that tags a protein’s mannose for lysosomal degradation I nstead of undergoing M6P, these should be degraded proteins follow the default secretory pathway to the cell exterior P rotein sorting→ The process of transporting proteins to its designated organelles by receptors on those organelles recognizing the protein sorting signals ○ Proteins are imported post-translation ○ T hese proteins are translated in the cytosol (does not undergo translocation) E.g. tail-anchored proteins P eroxisomes→ Single membrane organelles consisting of dense core of enzymes involved in the oxidation of different processes ○ E.g. Fatty acids are oxidized to generate hydrogen peroxide and energy The hydrogen peroxide is then broken down in the peroxisome by catalase ○ The precursor of peroxisomes comes from a vesicle from the ER membrane ○ Proteins enter or embed themselves into these organelles using signals P eroxisomal transport signal (PTS)→ Uses short signals to attract soluble proteinsto the peroxisomes (Ser-Lys-Leu) at the C-terminal M embrane peroxisomal transport signal (mPTS)→ Used to transport membrane proteinswith clusters of basic amino acids (e.g. arginine and lysine) on the C-terminus ○ P eroxins→ Receptors for peroxisomal proteins that binds to the signal in the cytosol and transports the proteins to the peroxisome This process requires ATP to be hydrolyzed Specific proteins can be studied using fluorescent microscopy: ○ Microscopes that allows us to modify the wavelength of light shined on the specimen and captured by the fluorescing specimen ○ F luorophores→ Fluorescent dyes that highlight certain region of a specimen The dye absorbs specific wavelength of light, causing its electrons to reach a high energy state Different dyes absorb different wavelengths W hen the electrons return to ground state, some of the absorbed energy is returned as a longer wavelength of light (colouration) This longer wavelength is what is captured by the fluorescent microscope ○ T he source of light is controlled using filters, where only the wavelength that excites the specimen is directed towards it ○ O nce the specimen fluoresces, thebeam-splitting (dichroic) mirroro nly allows the longer wavelength to be transmitted to the eyepiece ○ B efore entering theeyepiece, the light passes through asecond filterwhich captures specific fluorescent wavelengths which can then be observed through the eyepiece I mmunofluorescence→ The process of attaching a fluorescent molecule to an antibody that is specific to the protein of interest ○ Once the antibody attaches onto to the protein, we will be able to determine where our target protein is located due to fluorescence ○ A ntibody→ Proteins secreted by a type of white blood cells (B cells) that are made to defend against infection B cells carry a specific antibody which will react to its corresponding antigen (or protein) in the bloodstream D uring its encounter with the antigen, B cells undergo replication to create a large plasma cell that secretes many antibodies ○ Epitope→ The region(s) on an antigen which interacts with antibodies C ertain antigens can be large, where one side reacts with one antibody and its other side reacts with another antibody Therefore it has two epitopes P olyclonal antiserum→ The blood/antiserum obtained contains a collection of all the antibodies that react against a specific antigen The process of creating amonoclonal antibodies: ○ The immunized spleen cells of a mouse is suspended in a solution, along with myeloma cells (cancer cells) Myeloma cells are not able to grow in HAT medium ○ I n this suspension, the cells should be able to fuse together and become hybrid, followed by the addition of the cells into a selective HAT medium ○ In this medium, thehybridomas(hybrid cells) will continue to grow The unfused mice’s b-cells and cancer cells will die in HAT medium ○ Now, we have created an immortal cell that secretes the required antibody This is a monoclonal antibody because onlyone B-cellwas combined with the cancer cell
