Part 3 final exam The cytoskeleton assembly and dynamic structure Functions movement and structure o 1 Maintaining and directing cell structure o 2 Intracellular support Organelle movement Vesicle movement o 3 Spatial organization of cell o 4 Contractility and Mobility Actin myosin contraction Movement of cilia and flagella Chromosome movement Cell migration amoeboid movement Studying the cytoskeleton o Fluorescence Microscopy to see the fixed structure Immunofluorescence use of antibodies against cytoskeleton proteins Stain a fixed section of the cell with antibodies o The ones tagged with red proteins are directed against actin o Green tubulin o Blue nucleus Use of fluorescently tagged drugs that bind to cytoskeleton proteins E g phalloidin binds to actin filaments o Video enhanced light microscopy can detect presence of tubules and filament bundles and follow movement of vesicles in living cells but can t resolve details of structure o Electron Microscopy especially of freeze etched surfaces o Genetic engineering to study the FUNCTION of the cytoskeleton The development of genetically engineered organisms or cell lines that lack a cytoskeletal or motor protein knockout mutants has been enormously productive Mouse embryos that lack a motor protein called cytoplasmic dynein fail to develop beyond 8 days sue to dispersion and fragmentation of the Golgi complex suggesting a role for dynein in maintaining the structural integrity of the Golgi o Golgi breaks into vesicles in the absence of dynein 1 Cell lines or organisms can be engineered to over express an artificially constructed dominant negative form of a cytoskeletal protein Dominant negative proteins are non functional mutant proteins that compete with the native protein and inhibit its function o Interferes with the function of a normal protein o If you are able to develop and express this protein you can knock out the function of the protein you are targeting without knocking out the gene of the protein you are targeting Cells expressing the dominant negative protein show defects in the specific functions of the protein similar to those of knockout mutants Cells that are engineered to express small interfering RNA sequences siRNA s targeted to be complementary to the mRNA s of specific cytoskeletal proteins may also show defect in functions associated with those proteins The siRNA binds to the specific mRNA that has the complementary sequence promotes its destruction and silences the translation of the protein that the mRNA specifically codes for siRNAs can trigger the destruction of mRNA Cytoskeleton is formed from o 1 Microtubules o 2 Intermediate filaments o 3 Microfilaments Cytoskeleton can be dynamic changing or stable o Dividing fibroblasts o Stable Epithelial cells in the gut Mictotubules cell types o Large arrays of rigid microtubule bundles help maintain the overall shape of many 2 o Maintain the internal organization of cells Treatment with microtubule disorganizing drugs can seriously disrupt organelle distribution i e the Golgi is dispersed throughout the cell instead of being localized just outside the nucleus In plant cells microtubule bundles at the edge of the cytoplasm direct cellulose deposition in the neighboring cell wall by influencing the positioning of cellulose synthesizing enzymes o Important in maintaining the shape of very elongated cell extensions or processes i e Axons of nerve cells treatment with microtubule disorganizing drugs causes collapse of growing axons in the developing nervous system o Form the Mitotic spindles of dividing cells The core of cilia and flagella A network of rigid tubules that radiate through the cytoplasm of all eukaryote cells o Formed from alpha and beta tubulin Globular proteins approx 50kDa M W o Structure A rigid tube whose wall is made from approx 13 protofilaments composed of alternating alpha and beta tubulins Approx 25 nm in diameter and up to a micron or more in length The filament is polarized with a and a end end is composed of a row of beta tubulins grows more rapidly end is composed of a row of alpha tubulins o Proteins that bind to microtubules 1 Motor proteins kinesins and dyneins 2 MAPS microtubule associated proteins proteins that cross link MT s or regulate their assembly MAP2 its long projecting arm creates bundles of widely spaced Binding of MAPS to microtubules is often regulated by their microtubule arrays phosphorylation 3 o The tau protein a protein that regulates microtubule assembly in neurons Overphosphorylation of tau may be involved in creating the tangled neuronal structures observed in Alzheimer s Diseasepatients o Drugs that affect microtubules o Assembly prevents microtubule assembly Nocodazole Taxol Both of these drugs are anti mitotic prevent cell division since they disrupt prevents microtubule disassembly the assembly disassembly of microtubules that are needed to allow chromosomes to separate Taxol is used for chemotherapy since it will prevent division of tumor cells but also prevents mitosis in other cells hair follicles hair loss Assembly of microtubules both in vitro and in vivo begins with the creation of dimers between alpha and beta tubulin Dimerization requires that a GTP molecule is bound to the beta tubulin The dimers then associate into microtubule fragments and then elongate to form microtubules The GTP is hydrolyzed some time after the dimer is incorporated into a microtubule leaving GDP s still attached to tubulin subunits in the assembling microtubule Tubulin assembly can be achieved in cell homogenates in a test tube in vitro dead organisms Needs alpha tubulin beta tubulin and the addition of Mg2 GTP EGTA binds Ca2 an inhibitor of assembly Assembly disassembly can be produced by raising lowering Can be sped up by adding existing microtubule fragments nucleation temperature occurs on the fragments o Thus nucleation is the rate limiting step of assembly During periods of rapid growth of the end most tubulins present still have GTP attached creating a GTP cap with a different structure from the end where GDP predominates This cap stabilizes the end 4 Dynamic instability of the microtubule is the ebb and flow of microtubule length during a cell s activities The loss of a GTP cap can result in catastrophic shrinkage of the length and followed possibly by regrowth dynamic instability The growth or shrinkage of microtubules can be regulated by altering the balance of addition and removal of tubulin dimers MAPS that cap
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