Bio 401 L7 cytoskeleton 2024F.pptx

Bio 401 Cell Biology with Lab L7 Cytoskeleton Instructor: Dr. Joel Parker Office: Hudson 327 Phone #: 518-564-5279 Office hours: Wed 10am to noon, and by appointment E-mail: [email protected]  The...

Bio 401 Cell Biology with Lab L7 Cytoskeleton Instructor: Dr. Joel Parker Office: Hudson 327 Phone #: 518-564-5279 Office hours: Wed 10am to noon, and by appointment E-mail: [email protected]  The Thecytoskeleton has multiple cytoskeleton has multiple functions functions Gives the cell its structure Capacity to move or alter its shape Organisation and movement of organelles Transport of proteins and vesicles Actin in red Microtubules in green Nucleii in blue Comassie blue stain for proteins Shows the network inside the cell More on Organelles: Cytoskeleton: the matrix Oriented polarity, No polarity, just (centrisome to trafficking, structural outside), polarity, structure, trafficking, movement structure, movement Figure 18.1 Overview of the physical properties and functions of the three cytoskeletal systems in animal cells.  Components of the cytoskeleton Decreasing abundance Microfilaments (actin filaments) Intermediate Filaments Microtubules (tubulin) Microfilaments and microtubules are made up of subunits that can rapidly ssemble and disassemble ntermediate filaments are made of more stable fibrous protein subunits Figure 17.4 Examples of microfilament-based structures. Figure 17.3 Regulation of cytoskeleton function by cell signaling.  Structures of monomeric G-actin and F-actin filaments. domains with a central cleft where a divalent cation (Mg 2+ ) and nucleotide bind G-actin when F- actin when in a filament, globular, not in a polymerized form polymer  Myosin S1 decoration demonstrates the polarity of an actin filament. Chp 13.1 in Cooper used the old pointed and barbed terms. Actin subunits undergo cycling involving ATP and ADP G-actin Globular form of actin has 1 tightly bound Ca2+ and a non-covalently bound ATP G-actin can polymerise to form filamentous F-actin ATP is hydrolysed during the polymerisation but energy is not essential  (stable filaments added) Spontaneous formation Adding filaments Below Cc- andCc+, the filament dissembles or fails to form Above both Cc- and Cc+, filament grows at both ends with increasing concentration of monomer Below Cc- and above Cc+, tread milling, losing actin at the minus end, gaining at the positive end Treadmilling Figure 17.10 Actin treadmilling.  Figure 17.8 Determination of filament formation by actin concentration. A constant pool of available monomer, excess makes the filament grow Adding monomer  Regulation of actin polymerisation lin and thymosin levels can modulate the rate of microfilament synthesis Figure 17.11 Regulation of filament formation by actin-binding proteins. 1) Profilin accelerates binding of ATP to G- actin to speed up the process 2) Cofilin destablizes the minus end accelerating the process 3) Thymosin-beta 4 sequesters G-actin to slow down the process Figure 17.12 Capping proteins. ARP2/ARP3 mediated nucleation of actin polymerisation Arp2 and Arp3 are similar to G actin but cannot polymerise An Arp2/3 complex can act as a primer for actin polymerisation The arp2/3 complex can bind microfilaments (MF) resulting in branching of the MF network Regulation of microfilament association Filamin homodimers crosslink microfilaments forming a gel-like network Figure 17.13 Actin nucleation by the formin FH2 domain.  Microfilaments (main points) 1) One of the three types of cytoskeleton that provide structure, movement, and organization. 2) Microfilaments are polar (have direction) 3) Formed from actin dimers binding to ATP and ADP 4) Microfilaments are dynamic, they grow and contract 5) They are dynamic and grow or dissemble from both ends with differing concentrations of monomers 6) Actin filaments can treadmill 7) Monomer concentration is regulated to control  Microtubules Found in animal and plant cells α-tubulin and β-tubulin High degree of sequence similarity semble to form a 110kDa heterodimer which acts as the polymerising subunit lymerisation forms a tubular structure 25nm in diameter with a 14nm ternal pore α-Tubulin has a bound molecule of GTP, that does not hydrolyze. -Tubulin may have bound GTP or  GDP. Figure 18.3 Structure of tubulin dimers and their organization into microtubules. Figure 18.4 Singlet, doublet, and triplet microtubules. Figure 18.5 Microtubules are assembled from microtubule organizing centers (MTOCs). Figure 18.6 Structure of centrosomes.  Microtubule formation ntrosome is the major Microtubule Organising Center (MTOC) of animal Mix of proteins including the  -tubulin ring complex that nucleates the ubule growth. This matrix is organised by a pair of centrioles. plants microtubules form at multiple sites throughout the cell Isolated centrosome supplied with α and β tubulin Dynamic instability depends on the presence or absence of a GTP- -tubulin cap. The beta tubulin on the + , growing end, must have GTP bound or else it falls off. Over time, the GTP bound to beta tubulin becomes GDP and the microtubule falls apart.  Regulation of microtubule assembly Polymerisation of free tubulin subunits is energetically favourable min binds 2 αβ tubulin dimers thereby reducing the free pool and reducing tubule elongation hydrolysis will catch up with subunit addition resulting in loss of the GTP cap ition from the growing state to the shrinking state Figure 18.9 Dynamic instability of microtubules in vitro. Figure 18.15 Proteins that destabilize the ends of microtubules.  Stabilization of microtubules Microtubule Associated Proteins (MAPs) – allows crosslinking of MTs Can also mediate interaction with other cellular components Tau overexp. Regular spacing Closer arrangement of MTs Figure 16-41 Molecular Biology of the Cell (© Garland Science 2008)  Microtubules (main points) 1) One of the three types of cytoskeleton that provide structure, movement, and organization. 2) Microtubules are polar (have direction) 3) Formed from dimers binding to GTP and GDP 4) Microtubules are dynamic, they grow and contract 5) They are oriented with respect to the MTOC and the key source of orientation for intracellular trafficking and movement 6) MAP’s are key functional components for stability and function of microtubules. Intermediate Filaments Non-polarized Structural 5 basic classes, two shown here, Laminin in blue and keratin in red  Intermediate filaments ous polypeptides believed to assemble side by side to give rise to a filament h high tensile strength really known how the polypeptides are arranged in the filament be as a trimer with regions of α-helix and non-helical regions. -helical regions may stabilise the filament and interact with other mponents of the cytoplasm