CELL BIOLOGY - CLUTCH CH CYTOSKELETON AND CELL MOVEMENT.

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2 CONCEPT: OVERVIEW OF THE CYTOSKELETON The cytoskeleton is an intricate of protein filaments that extend throughout the cytoplasm It is a highly organized and dynamic structure (constantly moving and responding to the environment) There are three main components of the cytoskeleton - Intermediate filaments: Provide great tensile strength to the cell and nucleus - Acts as a protective cage for DNA - Microtubules: Provide support and internal framework to the cell - Creates mitotic spindle in cell division; Also forms cilia and flagella - Actin Filaments (microfilaments): Provides the plasma membrane with strength and shape - Allows for cell movement EXAMPLE: Actin is labeled in red, and microtubules are labeled in green Each component is created through joining of small subunits noncovalently linked together - Protofilaments are long string of subunits joined end to end - Can twist around other protofilaments to form helical lattice - Nucleation is the initiation process of assembling subunits requires an initial aggregation step Page 2

3 EXAMPLE: Nucleation of actin PRACTICE 1. Which of the following is not a component of the cytoskeleton? a. Intermediate filaments b. Actin filaments c. Nucleation d. Microtubules Page 3

4 2. What is the name of the initiation process that beings to assemble the subunits of the cytoskeleton? a. Cytoskeleton Initiation b. Support Initiation c. Nucleation d. Protofilament formation Page 4

5 CONCEPT: INTERMEDIATE FILAMENTS Intermediate filaments are cellular components that provide the cell with great strength Intermediate filaments are the most durable cytoskeletal filaments - They allow the cell to withstand mechanical stress - Strength provided by long intermediate filament strands that are wrapped together (like a rope) Intermediate filaments are anchored to the plasma membrane and the Intermediate filaments are formed by: - Individual subunits that link together to form one filament - Two filaments form dimers by binding via the alpha helical domain on the individual subunits - Two dimer sets arrange themselves in opposite directions (anti-parallel) to form tetramers - Two ends are the same Lack of or mutated forms of intermediate filaments can cause severe diseases - Ex: ALS, progeria EXAMPLE: Intermediate Filaments Page 5

6 There are four classes of intermediate filaments Classes Keratin Vimentin and Vimentin-related Neurofilaments Nuclear lamins Function Distributes stress in epithelial cells (like skin) Forms network from nucleus into the cell periphery Support the structure of the neuron Support nucleus; disassociate and re-form during cell division PRACTICE: 1. Which of the following is not a class of intermediate filaments? a. Nuclear lamins b. Neurofilaments c. Vimentin d. Integrins Page 6

7 2. Two intermediate filament dimers are arranged in which of the following ways to create a tetramer? a. Parallel b. Anti-parallel c. End-to-End Page 7

8 CELL BIOLOGY - CLUTCH CONCEPT: MICROTUBULES Microtubules are cytoskeletal elements that act as cellular for moving vesicles and organelles Tubulin is the subunit that makes up microtubules - Each tubulin subunit is a dimer composed of an alpha-tubulin and a beta-tubulin - Position of the subunits provides tubulin with polarity (plus end = beta and minus end = alpha) - Tubulin dimers add more quickly to the plus end Microtubule associated proteins bind to microtubules and stabilize them against disassembly EXAMPLE: Microtubule dimers Tubulin is attached to GTP which effect microtubule 1. Protofilaments (small aggregates of tubulin) form at microtubule-organizing centers in the cell (nucleation) 2. Tubulin polymerization occurs, which caused by adding to a growing molecule at either end 3. Tubulin dimers hydrolyze GTP (T form) to GDP (D form) - BUT speed of hydrolysis results in growth or instability - Hydrolyzed slowly: If GTP is hydrolyzed to GDP after the next dimer is added the filament will grow - A GTP cap is formed by attaching multiple tubulin-gtps without immediately hydrolyzing them - Hydrolyzed quickly: if GDP is hydrolyzed before the next dimer is added it will destabilize the microtubule Page 8

9 EXAMPLE: Tubulin polymerization and depolymerization GTP Protofilaments Polymerization GDP + end GTP cap - end Depolymerization Two terms describe the addition and destabilization of microtubules Dynamic instability is when a microtubule end switches between polymerization and depolymerization Treadmilling is when subunits are recruited to the plus end, and shed from the minus end EXAMPLE: Dynamic instability GTP growth GDP rescue catastrophe shrinkage Page 9

10 Microtubules and Cell Division Centrosomes are responsible for organizing microtubule during cell division Contains centriole pairs which act as a nucleation site for microtubule growth - Tubulin dimers are added with minus end towards the centrioles, and plus end towards cytoplasm EXAMPLE: Centriole pairs Page 10

11 PRACTICE: 1. Under which condition is a GTP cap formed during microtubule formation? a. If the microtubule end is hydrolyzed slowly b. If the microtubule end is hydrolyzed quickly 2. True or False: Treadmilling is when a microtubule end switches from polarization to depolarization. a. True b. False Page 11

12 3. A single tubulin subunit is composed of which of the following components? a. An alpha tubulin b. A beta tubulin c. A dimer of alpha and beta tubulin d. A tetramer of alpha and beta tubulin Page 12

13 CONCEPT: KINESINS AND DYNEINS Cells transport molecules, vesicles, and organelles throughout the cells on microtubules There are two types of cellular - Brownian movement is random thermal motions - Salutatory movement is jerky, stepwise movement in a single direction Motor proteins use energy from ATP hydrolysis to transport molecules across a microtubule in a single direction - Kinesins move molecules towards the plus end (away from cell body) - Over 14 families, but kinesin I is the one we will use as an example - Dyneins move molecules towards the minus end (towards the cell body) - Interacts with dynactin protein to move cargos over long distances There are multiple of motor proteins - Each has a different speed of movement depending on the cell type in which it s expressed EXAMPLE: Kinesins and Dyneins Dyneins Kinesins - + microtubule Motor proteins have a specific They function as dimers with two globular heads and a single tail - The two heads bind to microtubules in one orientation, and hydrolyze ATP to move along the microtubule - The tail attaches to vesicles, cargo, or organelles The heads go through repeated cycles of ATP hydrolysis to continually bind - Processive movement is movement that occurs for long distances without falling off (motor proteins) Page 13

14 EXAMPLE: Movement of kinesin along a microtubule Page 14

15 PRACTICE: 1. True or False: Kinesins move molecules away from the cell body a. True b. False 2. Where do motor proteins get the energy to move molecules throughout the cell? a. ATP b. GTP c. Breakdown of H2O d. Breakdown of sugars Page 15

16 3. Which of the following motor protein structures directly attaches to the cargo to transport it throughout the cell? a. One globular head b. Both globular heads together c. The motor protein tail d. The microtubule Page 16

17 CONCEPT: CILIA AND FLAGELLA There are two main organelles responsible for cell movement Multiple cilia are found on the plasma membrane and beat back and forth - Beating results in fluid movement or in propelling cells through fluid A single flagellum is found on the plasma membrane, and movements in a wavelike beating pattern - Results in propelling sperm or protozoa cells EXAMPLE: Flagellum vs. Cilia Flagellum Spins like a propellar Cilia Beats back and forth Cilia and Flagella have a similar structure Microtubules are arranged in a 9 +2 axoneme - Outer doublets: There are 9 doublets, equally spaced around the outside - Central pair: There are 2 doublets, placed in the center - Axoneme includes the microtubules and all associated proteins Each microtubule filament of each doublet is - A tubule has 13 microtubule protofilaments - B tubule has 10 or 11 microtubule protofilaments and fuses to the A tubule Connecting each of the 9 doublets together occurs through a variety of structures Interdoublet links connects two adjacent doublets together - Nexin is the protein responsible for this connection Page 17

18 EXAMPLE: A basal body is the region out of which the microtubules grows - Similar to a centriole - Composed of 9 triplets of microtubules Movement The sliding microtubule model describes how cilia and flagella Dynein is the motor protein responsible for this movement - Dynein bind to B tubules with its head groups - It move towards the minus end on B tubules with energy from ATP hydrolysis - As it moves towards the minus end on the B tubule It slides the A tubule down (bending) Sliding microtubule model is a model of movement based on core bending due to sliding microtubules Intraflagellar transport is the movement of molecules to and from the tips of the flagella Page 18

19 EXAMPLE: Sliding microtubule model Dynein Dynein Page 19

20 PRACTICE: 1. Microtubules are arranged in which of the following arrays? a b c d What is the name of the structure from which cilia and flagellum grow? a. Outer doublet b. Nexin c. Basal Body d. Central Pair Page 20

21 3. The structure of the basal body differs from the structure of the cilia in which of the following ways? a. It has a 9+3 structure instead of a 9+2 structure b. It is composed of 9 triplets of microtubules instead of the 9+2 structure c. It has an 8+2 structure instead of a 9+2 structure d. It is composed of only A tubules Page 21

22 CONCEPT: MICROTUBULES AND CELL DIVISION Microtubules are extremely important in cell division Many types of microtubules are responsible for the cell during division - Kinetochore microtubules attach to condensed chromosomes at centromeres - Chromosomal microtubules connect chromosomal ends to chromokinesin - Polar microtubules do not attach to chromosomes, but stabilize other microtubules - Astral microtubules extend outward from centrosomes to cell periphery EXAMPLE: Types of microtubules Astral Microtubules Polar Microtubules Kinetochore Microtubules The mitotic spindle and contractile ring are formed by microtubules during cell division Mitotic spindle is responsible for segregating replicated chromosomes into different daughter cells - Takes an hour for cellular microtubules to be disassembled and reorganized to form mitotic spindle Contractile ring is responsible separating the two daughter cells after division EXAMPLE: Mitotic spindle created by microtubules (Green) Page 22

23 Microtubules are important for each of mitosis Interphase: microtubules are long and stretched throughout the cell Prophase: microtubules are moved to opposite sides of the cell and form mitotic spindles Metaphase: Microtubules organize chromosomes in center of cell Anaphase: Sister chromatids separate and move to opposite poles Motor proteins are responsible for many of the microtubule movement in mitosis EXAMPLE: Microtubules during Mitosis Page 23

24 PRACTICE: 1. True or False: Microtubules are rarely used during cell division. a. True b. False 2. Which of the following microtubules attach to centromeres? a. Kinetochore b. Chromosomal c. Polar d. Astral Page 24

25 3. Which of the following microtubules do not attach to chromosomes? a. Kinetochore b. Chromosomal c. Polar Page 25

26 CONCEPT: ACTIN FILAMENTS Structure Actin filaments (microfilaments) are cytoskeletal elements crucial for cell movements G actin subunits are monomers that make up the larger actin filaments - F actin filaments are composed of two strands of G actin wound around each other - There are many types of actin (alpha = muscle; beta= nonmuscle; gamma = nonmuscle) Position of actin subunits provide to the F actin filaments - Minus end (pointed end) and plus end (barbed end) EXAMPLE: Actin filaments polymerize to that of microtubules G actin monomer are added to each end - However, addition occurs faster at the plus end than the minus end Each time a G actin monomer is added it hydrolyzes ATP shortly after addition - Hydrolyzed slowly: promotes filament growth - Hydrolyzed quickly: destabilization results in loss of actin polymers (minus end) Also undergoes dynamic instability and treadmilling - Dynamic instability is the rapid switch from growth to shrinkage at plus end Page 26

27 - Treadmilling is the gain of monomers at the plus end and loss of monomers at the minus end EXAMPLE: Page 27

28 Associated Proteins and Organization Many proteins are associated with actin to assist in, and function Arp2/3 complex and formins assist in nucleation which controls where actin filaments are formed ADF/Cofilin binds to actin and enhances disassociation of ADP-actin - Profilin reverses this action, and stimulates addition of actin monomers into filaments - Hydrolyzed quickly: destabilization results in loss of actin polymers (minus end) Proteins help regulate actin in many different ways - Ex: Regulate polymerization, cap actin filaments, crosslink, sever, bundle, attach actin EXAMPLE: Arp2/3 promoting actin filament formation The cell organizes actin in a few distinct ways Actin bundles form when actin filaments are cross linked into closely packed parallel arrays Actin networks form with actin filaments are cross linked into orthogonal arrays - 3D meshwork is formed with characteristics like that of a semisolid Cell cortex is composed of actin filaments and actin-associated proteins and lies beneath the plasma membrane Microvili are fingerlike extensions of the plasma membrane that are involved in absorption (made of actin) - Cells can have a brush border which is a layer of microvilli on the cell surface Page 28

29 EXAMPLE: Actin filaments supporting microvilli on the surface of plasma membrane Page 29

30 PRACTICE: 1. Which of the following proteins are associated with actin nucleation? a. Arp 2/3 b. ADF c. Profilin d. Integrin 2. True or False: Actin monomers are added to both the minus end and the plus end of a growing actin filament? a. True b. False Page 30

31 3. Which of the following terms describes the addition of monomers at the plus end and the loss of monomers at the minus end? a. Dynamic instability b. Treadmilling 4. If ATP at the minus end is hydrolyzed quickly, what happens to an actin filament? a. The filament grows at the minus end b. The filament is destabilized at the minus end c. The filament grows at both the minus and plus ends d. The filament is destabilized at both the minus and plus ends Page 31

32 CONCEPT: ACTIN BASED NON-MUSCLE MOVEMENTS There are three types of actin based non-muscle cellular movements Type Cell crawling (protrusion) Chemotaxis Cytoplasmic streaming Definition Cell drags itself forward by crawling over surfaces (Ex: amoebas, white blood cells, neutrophils) Migrating cell respond to differing concentrations of a diffusible chemical Cytosol streams back and forth within the cell (ex: slime molds and plant cells) Cell crawling uses four steps to move cells across a surface 1. Protrusion: Cell pushes actin based protrusion out from it s moving surface (driven by actin polymerization) - Pseudopodia (amoeba) - Lamelipodium is the dense leading portion which has filopedia protrusions at the leading edge 2. Attachment: Cellular protrusions attach to the surface - Integrins are transmembrane proteins that adhere to the ECM or the surface on which the cell is crawling 3. Translocation: The cell drags itself forward using the attached areas an anchorage points 4. Detachment: The cells detach from the surface EXAMPLE: Pseudopodia extensions from an amoeba Page 32

33 PRACTICE: 1. True or False: Pseudopodia are used by ameobas for cell crawling. a. True b. False 2. Which of the following proteins are used so that the cell can attach to the surface on which it is crawling? a. ECM proteins b. Filaments c. Filopedia d. Integrins Page 33

34 CONCEPT: ACTIN AND SKELETAL MUSCLE CONTRACTIONS Skeletal muscle contractions depend on between actin and myosin Myosin II are dimers with two globular ATPase heads and a long coiled-coil tail that extends outwards - Myosin filaments are formed by clusters of myosin II - Myosin filaments look like a double headed arrow Two myosin filaments bind to each filament - Each binds in opposite orientations, moving them in opposite directions EXAMPLE: Myosin Skeletal muscle has a distinct which allows for muscle contraction Myofibrils are cylindrical bundles of myosin and actin Sarcomeres are tiny contractile units that make up a myofibril - A band (dark band): composed of myosin (thick filaments) - H zone: lighter region of A band where myosin is not superimposed with actin - M line: Disc in the middle of the sarcomere - I band (light band): composed of actin (thin filaments), but no myosin - Z line (disc): Ends of the sarcomere EXAMPLE: Sarcomere Page 34

35 Steps to a Contraction Skeletal muscle contraction is caused from the shortening of sarcomeres 1. Myosin heads bind to actin 2. ATP hydrolysis results in the myosin head binding tightly, undergoing a conformational change, and moving - Tropomyosin is a protein that normally covers the actin binding site - Calcium binding to troponin molecules alter the structure of tropomyosin revealing the binding site 3. Cross-bridge forms, which is an overlap between thin and thick filaments (actin and myosin) - I band and H-zone shorten so the Z lines come closer together - All the band lengths stay the same actin just slides passed myosin to result in sarcomere shortening 4. ATP binds, which disassociates the cross bridge and it returns to its relaxed state EXAMPLE: EXAMPLE: Troponin and tropomyosin Page 35

36 PRACTICE: 1. Which of the following is not a structure of the sarcomere? a. A band b. H zone c. Z line d. U zone 2. Which of the following structures is composed of actin, but no myosin? a. A band b. M line c. I band d. Z line Page 36

37 3. When calcium binds to troponin, what happens to tropomyosin? a. It binds and covers the actin binding site b. It is removed from the actin binding site c. It creates a crossbridge structure d. It triggers ATP hydrolysis and myosin movement 4. True or False: When a cross-bridge structure is formed during a muscle contraction, the band lengths shorten and contract. a. True b. False Page 37