CHAPTER 9 The Cytoskeleton and Cell Motility Introduction

CHAPTER 9 The Cytoskeleton and Cell Motility

Introduction • The cytoskeleton is a network of filamentous structures: microtubulues, microfilaments, and intermediate filaments.

Properties of cytoskeletal components

9. 1 Overview of the Major Functions of the Cytoskeleton (1) • The cytoskeleton has many roles: – Serves as a scaffold providing structural support and maintaining cell shape. – Serves as an internal framework to organize organelles within the cell. – Directs cellular locomotion and the movement of materials within the cell.

Structure and functions of the cytoskeleton

Overview of the Major Functions of the Cytoskeleton (2) • Provides anchoring site for m. RNA. • Serves as a signal transducer. • An essential component of the cell’s division machinery.

9. 2 Study of the Cytoskeleton (1) • The Use of Live-Cell Fluorescence Imaging – Can be used to locate fluorescently-labeled target proteins. – Molecular processes can be observed (livecell imaging). – Used to reveal the location of a protein present in very low concentrations.

Applications using fluorescence imaging

Study of the Cytoskeleton (2) • The Use of In Vitro Single-Molecule Assays – They make possible to detect the activity of an individual protein molecule in real time. – Can be supplement with atomic force microscopy to measure the mechanical properties of cytoskeletal elements.

Using video microscopy to follow activities of molecular motors

9. 3 Microtubules (1) • Structure and Composition – Microtubules are hollow, cylindrical structures. – The microtubule is a set of globular proteins arranged in longitudinal rows called protofilaments. – Microtubules contain 13 protofilaments. – Each protofilament is assembled from dimers of α- and ß-tubulin subunits assembled into tubules with plus and minus ends.

The structure of microtubules

Microtubules (2) • Microtubule-Associated Proteins (MAPs) – MAPs comprise a heterogeneous group of proteins. – MAPs attach to the surface of microtubules to increase their stability and promote their assembly. – MAPs are regulated by phosphorylation of specific amino acid residues.

MAPs

Microtubules (2) • Microtubules as Structural Supports and Organizers – The distribution of microtubules determines the shape of the cell. – Microtubules maintain the internal organization of cells.

Microtubules (3) • Microtubules as Structural Supports and Organizers – Microtubules function in axonal transport. – Microtubules play a role in axonal growth during embryogenesis.

Microtubules (4) • Microtubules as Structural Supports and Organizers – In plant cells, microtubules help maintain cell shape by influencing formation of the cell wall.

Microtubules (5) • Microtubules as Agents of Intracellular Motility – They facilitate movement of vesicles between compartments. – Axonal transport: • Movement of neurotransmitters across the cell. • Movement away from the cell body (anterograde) and toward the cell body (retrograde). • Mediate tracks for a variety of motor proteins.

Axonal transport

Axonal transport

Visualizing axonal transport

Microtubules (6) • Motor Proteins that Traverse the Microtubular Cytoskeleton – Molecular motors convert energy from ATP into mechanical energy. – Molecular motors move unidirectionally along their cytoskeletal track in a stepwise manner. – Three categories of molecular motors: • Kinesin and dynein move along microtubule tracks. • Myosin moves along microfilament tracks.

Microtubules (7) • Kinesins – Kinesin—member of a superfamily called KLPs (kinesin-like proteins). – A kinesin is a tetramer of two identical heavy chains and two identical light chains. – Each kinesin includes a pair of globular heads (motor domain), connected to a rod-like stalk. – Kinesin is a plus end-directed microtubular motor based on its movement.

Kinesin

Microtubules (8) • Kinesins (continued) – They move along a single protofilament of a microtubule at a velocity proportional to the ATP concentration. – Movement is processive, motor protein moves along an individual microtubule for a long distance without falling off. – KLPs move cargo toward the cell’s plasma membrane.

Kinesin-mediated organelle transport

Microtubules (9) • Cytoplasmic Dynein – responsible for the movement of cilia and flagella. – Cytoplasmic dynein – Huge protein with a globular, force-generating head. – It is a minus end-directed microtubular motor. – Requires an adaptor (dynactin) to interact with membrane-bounded cargo.

Cytoplasmic dynein

Cytoplasmic dynein

Microtubules (10) • Microtubule-Organizing Centers (MTOCs) – MTOCs – specialized structures for the nucleation of microtubules. – Centrosome – structures responsible for initiating microtubules in animal cells. • It contains two barrel-shaped centrioles surrounded by pericentriolar material (PCM). • Centrioles are usually found in pairs.

The centrosome

The centrosome

Microtubules (11) • Centrosomes (continued) – Responsible for initiation and organization of the microtubular cystoskeleton. – Microtubules terminate in the PCM.

Microtubules (11)

Microtubules (12) • Basal Bodies and Other MTOCs – Basal body – structure where outer microtubules in a cilia and flagella are generated. – Plant cells lack MTOCs and their microtubules are organized around the surface of the nucleus.

Microtubules (13) • Microtubule Nucleation – MTOCs control the number of microtubules, their polarity, the number of protofilaments, and the time and location of their assembly. – The protein -tubulin is found in all MTOCs and is critical for microtubule nucleation.

The role of -tubulin in centrosome function

Microtubules (14) • The Dynamic Properties of Microtubules • There are four distinct arrays of microtubules in a dividing plant cell: – Widely distributed throughout the cortex. – Making a single transverse band. – In the form of a mitotic spindle. – As a phargmoplast assisting in the formation of the cell wall of daughter cells.

Four arrays of microtubules in a plant cell

Microtubules (15) • The Dynamic Properties of Microtubules – Newly formed microtubules branch at an angle of pre-existing microtubules. – The changes in spatial organization of microtubules are a combination of two mechanisms: • Rearrangement of existing microtubules. • Disassembly of existing microtubules and reassembly of new one in different locations.

Nucleation of plant microtubules

Nucleation of plant microtubules

Microtubules (16) • The Underlying Basis of Microtubule Dynamics – Insight into factors that influence microtubule assembly and disassembly came from studies in vitro. – GTP is required for microtubule assembly. – Hydrolysis of GTP leads to a replacement of bound GDP by new GTP to “recharge” the tubulin dimer.

Microtubule assembly in vitro

Structural cap model of dynamic instability