Cytoskeleton 1 (1) PDF

Cellular Biology & Homeostasis CYTOSKELETON Part 1 VP Summer 2023 Clara Camargo, DVM LEARNING OBJECTIVES 1. Briefly describe the cytoskeleton: characteristics and function 2. Describe the structure and function of:  Actin filaments (cell shape and movement, polymerization/depolymerization) THE...

Cellular Biology & Homeostasis CYTOSKELETON Part 1 VP Summer 2023 Clara Camargo, DVM LEARNING OBJECTIVES 1. Briefly describe the cytoskeleton: characteristics and function 2. Describe the structure and function of:  Actin filaments (cell shape and movement, polymerization/depolymerization) THE CYTOSKELETON how do cells maintain their shape? Cell Organelles 2 Cytoskeleton https://www.youtube.com/watch?v=tO-W8mvBa78 CYTOSKELETON THE CYTOSKELETON DETERMINES CELLULAR ORGANIZATION AND POLARITY A detailed network of protein filaments (intermediate, microtubules, actin) that extends throughout the cytoplasm. All three cytoskeleton filament systems must normally function collectively to give a cell its strength, shape, and ability to move. Importance  Cells need proper organization in space  Need to interact with each other  Need to interact with their environment CYTOSKELETON Facilitates the existence of special structures: • Microvilli - cellular membrane protrusion, increase surface area • Desmosomes - special adhesive protein complexes that help maintain mechanical integrity • Adherens Junctions - protein complexes that occur at cell-cell junctions • Apical and Basolateral Membranes - Apical (towards the lumen) Basolateral (away from lumen) The proteins that make up the filaments of the cytoskeleton can form polarized and selforganized structures that can be highly dynamic, allowing the cell to rapidly modify its structure and function under different conditions. CYTOSKELETON THE CYTOSKELETON ENABLES A CELL: • To organize and maintain its correct shape and structure (external/internal) • To resist mechanical deformation • To stabilize itself and its environment (by associating the cell to other cells and to its surrounding extracellular tissues) • To change its shape for movement and migration THREE DIFFERENT TYPES OF FILAMENTS COMPOSE THE CYTOSKELETON: Actin filaments Intermediate filaments Microtubules CYTOSKELETON CLASSIFICATION OF CYTOSKELETON COMPONENTS IMMUNOFLUORESCENCE STAINING DETECTION OF ACTIN FILAMENTS (MICROFILAMENTS) CYTOSKELETON CLASSIFICATION OF CYTOSKELETON COMPONENTS Photo: Wikipedia IMMUNOFLUORESCENCE DETECTION OF MICROTUBULES CYTOSKELETON CLASSIFICATION OF CYTOSKELETON COMPONENTS Photo: Nikon’s small world IMMUNOFLUORESCENCE DETECTION OF INTERMEDIATE FILAMENTS CYTOSKELETON CLASSIFICATION OF CYTOSKELETON COMPONENTS Microfilaments/ Actin filaments Actin filaments determine the shape of a cell and are necessary for cell locomotion 7 nm Microtubules 24 nm Intermediate filaments 8-12 nm Microtubules determine the positions of membrane-enclosed organelles, direct intracellular transport, and form the mitotic spindle Intermediate filaments provide mechanical strength CYTOSKELETON  Actin filaments and microtubules are built from subunits that are compact and globular.  Intermediate filaments are made up of smaller subunits that are elongated and fibrous All three filaments form helical assemblies of subunits that self-associate, using a combination of end-to-end and side-to-side protein contacts. CYTOSKELETON Actin Tubulin Intermediate filaments MICROFILAMENTS - ACTIN STRUCTURE & FUNCTION Found in eukaryotes cells, actin performs a wide range of functions in cells Essential for:  Mechanical support  Movements → cell crawling, engulfing, migrate, muscle movement  Cell shape, structure → microvilli ACTIN- STRUCTURE & FUNCTION Form a tough, but flexible framework  G-Actin subunits are compact and globular. Form a tight, right-handed helix called filamentous actin (F-actin) (+)-End • Actin-Filament (F-Actin) consists of 2 parallel protofilaments • Flexible structure (-)-End • Usually shorter than microtubules ACTIN- POLYMERIZATION Actin filaments can grow by adding more actin monomers at either end (-) or (+): o Nucleation o Steady State o AT P Elongation Each free actin monomer carries a tightly bound ATP o ATP bound actin has a higher affinity for the neighboring subunits and remains stable in the filament o ADP bound actin can more easily dissociate from the filament  Hydrolysis of ATP->ADP  Reduces strength of binding b/t monomers  Decreases polymer stability Accessory proteins regulate actin dynamics  Profilin – inhibits nucleation  Cofilin – accelerate depolymerization https://www.youtube.com/watch?v=VVgXDW_8O4U ACTIN – Polymerization and depolymerization Actin filaments can polymerize to grow or depolymerize ATP bound actin has a higher affinity for the neighbouring subunit and remains stable in the filament ADP bound actin can more easily dissociate from the filament https://www.youtube.com/watch?v=n9q7Imlnm8 ACTIN – STRUCTURE (Cell shape) Bundle-forming crosslinker (more rigid) Gel-forming crosslinker (networks) Actin filaments exist in different spatial arrays in cell Formation depends on actin crosslinking proteins (actinbinding proteins) o Fascin: linear bundles o Filamin: 3D gel-forming networks ACTIN – Microvilli support • Actin filaments are crosslinked by o Closely packed parallel arrays • Proteins involved in crosslink: o Small and rigid o Force filaments to align closely o Example is espin, villin, fimbrin • Can support the way microvilli project • Can also contract ACTIN- 3D Gel forming Gel-forming crosslinker Loosely crosslinked in a 3D like meshwork with the semisolid gel like properties Proteins involved in network: o Large and Flexible o Crosslink more perpendicular o Filamin ACTIN – Cell movement Actin crosslink and cell movement Ameba movement https://www.youtube.com/watch?v=TVXxJCAj4Ds&t=2s ACTIN – Cell movement Three main processes are known to be essential for movement and all involve ACTIN 1. The cell pushes out its protrusion at its ”front” or “leading edge” 2. These protrusions stick to the surface over which the cell crawls 3. The rest of the cell drags itself forward by traction Examples of cells that do this: 1. Carnivorous amoeba 2. Neutrophils (WBC) 3. Developing Axons in response to growth factors 4. Fibroblasts ACTIN - CELL DIVISION CYTOKINESIS: The part of cell division when the eukaryotic cell divides into 2 daughter cells Following completion of mitosis (nuclear division), a contractile ring consisting of actin filaments and myosin (II) divides the cell in two • Cell membrane will grow inward (cleavage) and pinches off ACTIN - ASSOCIATED PROTEINS → MOTOR PROTEINS Myosin, Kinesin & Dynein • Motor proteins that cause motion inside cells in association with parts of the cytoskeleton • All are ATPases • ATP → mechanical energy (transport, muscle movement, beating of cilia and flagella) Actin associates with myosin to form contractile structures All actin-dependent motor proteins belong to the myosin family ACTIN – Motor proteins: Myosin family Myosin I o 1 head and a tail o Transporting vesicles (tail can bind to) Myosin II o Two globular (ATPase) heads and coiled tail o Produces muscle contraction in most animal cells o In non-muscles cells: contractile bundles-stress fibers Myosin V o Cargo transporter (i.e. RNA, Vesicles, Organelles, Mitochondria) o Tether like keeping vesicles and organelles in the actin-rich periphery of cells ACTIN - Motor proteins: Myosin family All myosin heads interact with actin filaments ATP hydrolyzing motor to help it move ACTIN & MYOSIN IN MUSCLE Myofibril: contractile elements of the muscle cell - Consists of a chain of tiny identical contractile units called sarcomeres - Sarcomeres- highly organized assembly of 2 types: actin and myosin II filaments ACTIN AND MYOSIN – Muscle contraction Sarcomeres: Highly organized assembly of 2 types: actin and myosin II filaments →it’s a functional unit Contraction is caused by a simultaneous shortening of all the sarcomeres Caused by Actin filaments sliding past the myosin filaments ACTIN- Muscle contraction Sliding Filament Model: A process used by muscles to contract  Myosin heads interact with the actin filaments  The thin filament slides over the thick filament to cause tension  Depends on sequence of molecular events: Crossbridge cycling Muscle stimulated → Contraction → Myosin head walks along the actin filament This happens over and over again (repeated cycles of attachment and detachment) Sarcomere contracts ACTIN - Muscle contraction ACTIN - Muscle contraction ASSOCIATION OF TROPOMYOSIN AND TROPONINS WITH ACTIN FILAMENTS – Striated muscle Tropomyosin binds lengthwise along actin filaments and is associated with a complex of three troponins: • troponin I (TnI) • troponin C (TnC) • troponin T (TnT) In the absence of Ca2+, the tropomyosin-troponin complex blocks the binding of myosin to actin Binding of Ca2+ to TnC moves the complex, freeing the myosin-acting binding site, and allowing contraction to proceed ACTIN AND MYOSIN - MUSCLE CONTRACTION Signal from the nervous system Triggers an action potential o Electrical impulse → depolarize each myofibril → sarcoplasmic reticulum (main function is to store Calcium Ions) o Release Ca⁺ ⁺ o Ca⁺ ⁺ interacts with → Troponin complexTropomyosin ACTIN AND MYOSIN - MUSCLE CONTRACTION https://www.youtube.com/watch?v=BVcgO4p88AA ACTIN – Drugs affecting filaments ACTIN - Drugs affecting filaments Chemical compounds that stabilize or destabilize actin filaments are important tools in studies of the filaments’ dynamic behavior and function in cells. PHALLOIDIN It can be used for staining actin filaments  Binds to all variants of actin filaments in many different species of animals and plants (very specific)  Binds actin filaments and blocks depolymerization ALPHA-AMANITIN Main toxic component of the death cap mushroom  inhibits RNA-polymerase II