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Lecture 7

Lecture 7 and 8.docx

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Jane Mitchell

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Lecture 7 and 8 the cytoskeletonComposed of 3 diff filament classesDynamic structure found in eukaryotes o Provides structural transport positions organelles directs vesicular transport involved in locomotion flagella sperm cilia paramecium cell crawling sheets of membrane crawl to active site required for cell division involved in muscle contraction o Proteins are directed to specific place bw cytoskeletonThe cytoskeleton provides structural support RBC poked with laser tweezers returns to its original shape due to structural support provided by cytoskeletonIt withstands tears and allows them to bring back to original shapeVideo3 types of filaments form the cytoskeleton o Microfilaments actin proteinsDiameter59nmForm long filaments made of protofilaments that are twisted around each other o Intermediate filaments intermediate filament proteinsDiameter10nmThey are flexible and extensiveHas ropelike qualitieso Microtubules made of turbulent proteinsDiameter25nmIndividual protofilaments allow structure to be organized to microtubule organised centreTechniques used to study cytoskeleton o Immunoflorescence technique used to determine the location of proteins within cellCells are FIXEDIt is possible to locate more than one protein at a time and compare them o Antibody is used which binds specifically to protein of interest o Second antibody binds to the first antibody and is covalently tagged with fluorescent molecule o Fluorescence microscope is used to excite fluorescence molecule and visualise light emittedLight microscopy has resolution limit due to diffraction based on wavelength of light 250 nmElectron microscope electrons shorter wavelengthResolution 10 Angstroms 250x better resolutionDynamic structure electron microscope is not useful for this o For each cell motilitycrawling the actin filaments must rapidly assemble and disassemble at the leading edgeEg a neutrophil chasing clump of bacteriaEg Microtubulesmost interphase microtubules radiate from one microtubule organizing center which is located near nucleusthey are recognized to form bipolar mitotic spindles in dividing cellscytoskeletal filaments are composed of small soluble subunits that form polymers which allows them to be dynamicCan rapidly assemble and disassembleHow are these polymers constructed need to be strong so that they can have support functions within cellThey need to be flexible and easily disassemble and reassemble o Cytoskeletal filaments constructed of multiple chains Single chain polymers with Strong bonds Weak bonds Thermal stability High Low Dynamic filament Low HighMultiple subunits are assembled into bundles of protofilamentsindividual protofilaments associate with each other laterally o Strength of cytoskeletal filments is due to many weak bondsTogether they build a stronger filaments o Each individual bond doesnt need to be as strong and that allows the cyctoskeletal components to be both strong and dynamic o All actin filaments intermediate filaments and microtubules are all constructed of more than one protofilamentActin polymerisation in vitro experiments high concentration of actin subunits is added to the test tube under low salt conditionsWhen salt added it helps favour polymerase reaction subunits start to form up into filamentsThere is a lag time after addition of salt during this step an association bw 2 subunits might form that eventually break apartNucleus formed association of 3 or more subunits here bonds start to come stabilized and nucleus is at a position where more subunits can be incorporatedActin filaments start to growlinear growth by addition of proteins from soluble poolOnce filaments get to certain size reaches equilibrium where the net addition of soluble poolloss of filamentThere is no growth of filament after this critical concentration concentration of free filament subunits at which the filaments in solution will be at a steady state with soluble poolMicrotubules involved in intracellular transport radiate out from center of cell have motor proteins that move system giving them directionality structural support cell organization position organelles mitosis by forming spindle cell motility flagella and cilia
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