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Transcript
Introduction to MCB380 2008 • Instructor: Juliet Lee • Email [email protected] • Class website: http://web.uconn.edu/mcb380/ • Goal: To understand how the cytoskeleton, integrates and coordinates cell functions Whole organism Large – How do molecular scale events affect cellular and tissue level behavior or function? Tissue level – Major examples: Cell motility, cell division, signaling, cancer Cell level Molecular Monomers Small Filament 1. What is the nature of the cytoskeleton at the molecular level? The cytoskeleton is a dynamic structure • Cytoskeletal filaments can grow and shrink (or both) as subunits are assembled or disassembled, respectively. • The cytoskeleton can rapidly reorganize itself in response to its environment by means of different signaling mechanisms. • The structure and dynamics of the cytoskeleton is intimately linked to its function. 2. How does the cytoskeleton function in a whole cell? Cell movement Protrusion at front Protrusive force Adhesion formation Adhesive force Forward shift of cell body Contractile force Contractile force De-adhesion Retraction at rear Cell division • 1. The role of microtubules and motors in generating the forces necessary for: • Spindle formation in prophase • Chromosome alignment – metaphase • Separation of duplicated chromosomes – anaphase • Separation of daughter nuclei – telophase • Separation of daughter cells cytokinesis How is the cytoskeleton involved in the functioning of tissues or the whole organism ? Signaling: Regulation of mitosis and cell motility Loss of growth control Increased motility Cancer • Course structure: – – – – – Lecture period ~45 min. Workgroup problem or Presentation of a research paper Exams, part I (in class) part II (take home) Group problems/papers = 30% , exams = 70% of final grade • Lectures (PowerPoint)/ papers will be posted on the MCB380 website or HuskyCT (if more space is needed) Overview of the actin cytoskeleton • Where is it? – Methods of visualization, electron microscopy and fluorescence microscopy • What is it ? – Molecular composition • How is it assembled? – dynamics • What are its functions ? – Structure, – Many others… How was the cytoskeleton discovered? • The cytoskeleton was first identified by electron microscopy (EM). • 3 different filament systems were identified on the basis of their diameter and surface texture. • A major characteristic of the cytoskeleton is its insolubility in non ionic detergents - allowing observation and biochemical characterization. • A freeze-dried metal replica of a fibroblast, viewed by EM provides a very high resolution image of cytoskeletal elements. Why was (EM) important for the discovery of the cytoskeleton ? Resolving power of light microscope compared with EM 200 nm . The maximum resolution of a light microscope is ~ 200 nm The resolving power of the electron microscope is much higher - because the wavelength of electrons is much smaller than that of light ~ 1.5nm. Immunofluorescence shows the location of different cytoskeletal filaments Actin filaments • Intermediate filaments Microtubules Cytoskeletal proteins can be labeled with fluorescently tagged (conjugated) antibodies, which bind with high affinity and specificity. (Usually in fixed cells) – Can be done in living cells to investigate function of a specific filament system • Cytoskeletal proteins can be labeled with fluorescent molecules and injected into living cells - to see where they become localized The cytoskeleton is a filamentous polymer meshwork • The cytoskeleton consists of three different types of filament systems Actin, diameter 7-9 nm Sub unit = actin monomer • Each filament is a polymer built from small protein subunits, which are held together non covalently. Microtubules, 25 nm Sub unit = tubulin heterodimer • Note that: proteins are also polymers composed of amino acid monomers, BUT they are bound together covalently. Intermediate 10 nm Sub unit = α helical subunits Thermal stability of a single filament is increased by “side” binding with others • A single filament is not strong enough to avoid breakage from ambient thermal energy - because each monomer is linked NONcovalently • Lateral binding with other “protofilaments” increases the strength of the entire filament • Single monomers can be added or lost from ends more easily than the filament can be broken in the middle The cytoskeleton is multifunctional. The cytoskeleton can form permanent and transient structures Microvilli are permanent actin containing structures Ruffles on the surface of a moving cell are transient actin containing structures EM view of the cytoskeleton in a gut epithelial cell Permanent microtubule-containing structures • Didinium: A carnivorous ciliate • One protozoan eating another • ( Fig. 1-28, Alberts 3rd Ed.) The mitotic spindle is a transient microtubulecontaining structure