Chapter 9.1 Cytoskeleton, Cell Cycle, and Microtubules PDF

CHAPTER 9 – part 1 The Cytoskeleton, Cell Cycle and Microtubules Required reading: chapter 9- appropriate sections Chapter 14 – appropriate sections 9.1-1 9.1 | Overview of the Major Functions of the Cytoskeleton Properties of cytoskeletal...

CHAPTER 9 – part 1 The Cytoskeleton, Cell Cycle and Microtubules Required reading: chapter 9- appropriate sections Chapter 14 – appropriate sections 9.1-1 9.1 | Overview of the Major Functions of the Cytoskeleton Properties of cytoskeletal components 9.1-2 9.1 | Overview of the Major Functions of the Cytoskeleton Properties of cytoskeletal components 9.1-3 9.1 | Overview of the Major Functions of the Cytoskeleton Roles of cytoskeletal components 9.1-4 9.1 | Overview of the Major Functions of the Cytoskeleton Roles of cytoskeletal components 9.1-5  9.1 | Overview of the Major Functions of the Cytoskeleton Roles of cytoskeletal components: cytoskeletal changes during cell division Division of a polarized fibroblast cell Purple - Actin filaments Green - Microtubules Brown - DNA Cell polarity = spatial differences in shape, structure, and function within a cell Almost all cell types exhibit some form of polarity, which enables them to carry out specialized functions 9.1-6 Epithelial cells have apical-basal polarity the cytoskeleton and signaling 9.1-7 9.1-8 9.1-9  9.2 | Structure and Function of Microtubules Structure and Composition of Microtubules Microtubules are the largest of the cytoskeletal components of a cell There are two types of microtubules They are involved in a variety of functions in the cell 1) Cytoplasmic microtubules found in the cytosol and are responsible for a variety of functions: Maintaining axons (in nerve cells) Formation of mitotic and meiotic spindles Placement and movement of vesicles Maintaining or altering cell shape 2) Axonemal microtubules include the organized and stable microtubules found in structures specialized for movement such as: Cilia Flagella Basal bodies to which cilia and flagella attach The axoneme, the central shaft of a cilium or 9.1-10 flagellum, is a highly ordered bundle of MTs  9.2 | Structure and Function of Microtubules Structure and Composition of Microtubules MTs are straight, hollow cylinders of varied length that consist of (usually 13) longitudinal arrays of polymers called protofilaments The basic subunit of a protofilament is a heterodimer of tubulin, one α-tubulin and one β-tubulin (globular proteins) These bind noncovalently to form an αβ-heterodimer, which does not normally dissociate 9.1-11 9.2 | Structure and Function of Microtubules Microtubule-Associated Proteins Microtubules typically contain additional Schematic diagram proteins, called microtubule-associated of a brain MAP2 proteins (or MAPs) molecule bound to the surface of a MAPs comprise a heterogeneous collection microtubule. of proteins with one domain that attaches to the side of a microtubule and another domain that projects outward as a tail MAPs generally increase the stability of microtubules and promote their assembly MAP activity is controlled by the addition and removal of phosphate groups from amino acid residues An abnormally high level of phosphorylation of one particular MAP, called tau, has been implicated in Alzheimer’s disease A variety of MAPs 9.1-12  9.2 | Structure and Function of Microtubules Structure and Composition of Microtubules Building block of a microtubule is a dimer of  tubulin and β tubulin (each 55,000 Da)  and β tubulin are two related globular proteins (40% amino acid identity) noncovalently bound together (they are not found as individual monomers in the cell) Each monomer has a GTP binding site GTP in the  monomer is physically trapped at the dimer interface It is never hydrolyzed or exchanged In the β tubulin monomer the nucleotide can be in either GDP or GTP form (and is both hydrolyzable and exchangable) 9.1-13  9.2 | Structure and Function of Microtubules Structure and Composition of Microtubules All the dimers in the MT are oriented the same way Because of dimer orientation, protofilaments have an inherent polarity The two ends differ both chemically and structurally minus(-) end and a plus (+) end 9.1-14  9.2 | Structure and Function of Microtubules Microtubules as Structural Supports and Organizers Microtubules resist forces that might compress or bend the fiber for mechanical support Distribution of microtubules helps determine the shape of that cell In cultured animal cells, microtubules extend in a radial array outward from near the nucleus, giving cells a round, flattened shape Microtubules of columnar epithelial Localization of microtubules in a cells are oriented with their long axis cultured mouse cell shown by parallel to the long axis to support the fluorescent anti-tubulin antibodies cell’s elongated shape Microtubules extend from the perinuclear region of the cell in a radial array and curve gradually as they conform to the shape of the cell 9.1-15  9.2 | Structure and Function of Microtubules Microtubules as Structural Supports and Organizers Microtubules also play a key role in maintaining the internal organization of cells Treatment of cells with nocodazole or colchicine, which promote microtubule disassembly, can disperse the Golgi elements into separate Golgi stacks scattered throughout the cytoplasm When the drug is removed and microtubules reassemble, the Golgi membranes return to their normal position in the cell interior red = microtubules Green = Golgi 9.1-16  9.2 | Structure and Function of Microtubules Microtubules as Structural Supports and Organizers Green = cellulose synthase Red = microtubules 9.1-17 Motors and the cytoskeleton Kinesin – Anterograde MT motor Dynein – Retrograde MT motor Myosin is a third motor protein that carries organelles along actin fibers 9.1-18  9.4 | Motor Proteins: Kinesins and Dyneins 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 9.1-19  9.4 | Motor Proteins: Kinesins and Dyneins Motor Proteins Traverse the Microtubular Cytoskeleton Motor proteins move unidirectionally along their cytoskeletal track in a stepwise manner and undergo a series of conformational changes that constitute a mechanical cycle The steps of the mechanical cycle are coupled to the steps of a chemical cycle, which provide the energy necessary to fuel the motor’s activity and to move along the track The chemical cycle steps include the binding of ATP to the motor, the hydrolysis of ATP, the release of the products (ADP and Pi), and the binding of a new molecule of ATP Motor proteins have virtually no momentum and are subjected to tremendous frictional resistance from their viscous environment As a result, a motor protein comes to a stop almost immediately once energy input has ceased 9.1-20 9.4 | Motor Proteins: Kinesins and Dyneins Kinesins In vitro motility assays show that kinesin is a plus end-directed microtubular motor Each kinesin step is approximately 8 nm, the length of one tubulin dimer, and requires the hydrolysis of a single ATP molecule Kinesin movement: is proportional to the ATP concentration moves in a “hand-over-hand” mechanism is highly processive (can walk along a microtubule for considerable distances without falling off) 9.1-21 9.4 | Motor Proteins: Kinesins and Dyneins Cytoplasmic Dynein Kinesins and cytoplasmic dynein move similar materials in opposite directions over the same railway network microtubules may bind kinesin and dynein simultaneously Organelles may bind both kinesin and dynein simultaneously with one of them innactive 9.1-22 Movement along microtubules can be regulated Melanosome aggregation is via dynein Melanosome dispersed via kinesin 9.1-23  9.5 Microtubule Dynamics The dissociation rate of a GDP tubulin The Dynamic Properties of Microtubules dimer is much more rapid than the dissociation of a GTP tubulin dimer The process of alternating between growing and shrinking = dynamic instability 9.1-24  9.5 | Microtubule Organizing Centers (MTOCs) The function of a microtubule within a living cell depends on its location and orientation, which makes it important to understand why a microtubule assembles in one place as opposed to another When studied in vitro, the assembly of microtubules from αβ-tubulin dimers occurs in two distinct phases: a slow phase of nucleation, in which a small portion of the microtubule is initially formed, and a much more rapid phase of elongation Unlike the case in vitro, nucleation of microtubules takes place rapidly inside a cell, where it occurs in association with a variety of specialized structures called microtubule-organizing centers (or MTOCs) All MTOCs play similar roles in all cells: they control the number of microtubules, their polarity, the number of protofilaments that make up their walls, and the time and location of their assembly. The best studied MTOC is the centrosome 9.1-25  14.1 | The Cell Cycle: Phases of the Cell Cycle The cell cycle is the series of stages that a cell goes though. It consists of M phase (mitosis) and interphase ((G1, S, G2) M phase includes the process of mitosis and cytokinesis Mitosis lasts ~ hour or so Interphase (G1, S, G2) constitutes the majority of the cell cycle (90%) and lasts longer than the M phase; it may extend for days, week, or longer but average for mammalian cells in ~ 24 hours 9.1-26 14.1 | The Cell Cycle Phases of the Cell Cycle 9.1-27  14.2 | Overview of M Phase: Mitosis and Cytokinesis Mitosis is generally divided into five stages: prophase prometaphase metaphase anaphase telophase Each is characterized by a particular series of events. Each of these stages represents a segment of a continuous process. 9.1-28 9.5 | Microtubule Organizing Centers (MTOCs) Centrosomes Made up of a complex of proteins not all of which have been identified γTURCs:Tubulin Ring complex where nucleation occurs Centrioles: short cylinders of modified microtubules 9.1-29 The growth of microtubules occurs by addition of subunits at the plus end of the polymer away from the centrosome the minus end is associated with the centrosome Genetic defects in centrosome-associated proteins cause microcephaly, presumably because neuron proliferation and migration is sensitive to loss of centrosome function 9.1-30 Centrioles: short cylinders of modified microtubules 9.1-31 Nucleation of a microtubule begins with γ-tubulin at the minus end nucleation itself is initiated by γTURCs: Tubulin Ring complex The γ-TuRC is a helical array of γ- tubulin subunits where αβ-tubulin dimers assemble 9.1-32  14.2| Three types of microtubules The mitotic spindle contains three classes of microtubules Kinetochore microtubules: are connected to chromosomes (after first “finding them via a kinetochore on the chromosome) Astral microtubules: project toward the cell cortex and interact with it thereby orienting the spindle of division Polar microtubules: interact with microtubules from the opposite pole of the cell 9.1-33  14.2 | Prophase Formation of the Mitotic Chromosome Protein synthesis stops Internal membrane systems that are normally associated with microtubules disperse Endocytosis and exocytosis stop Each centrosome (that divided in S phase) forms an MTOC and nucleation and elongation of microtubules begins How do the microtubes reach the chromosomes in the nucleus? 9.1-34  14.2 | Prophase The Dissolution of the Nuclear Envelope and Partitioning of Cytoplasmic Organelles at the end of prophase The three major components of the nuclear envelope: nuclear pores nuclear lamina (structural and composed of intermediate filaments) Nuclear membranes are disassembled in separate processes The integrity of the nuclear membranes is first disrupted mechanically as holes are torn into the nuclear envelope by cytoplasmic dynein molecules associated with the outer nuclear membrane Phosphorylation of human lamin causes de-polymerization of the intermediate filaments in the nuclear lamina mitochondria, lysosomes, and peroxisome remain relatively intact Golgi is either absorbed by the ER or 9.1-35 fragmented and partitioned  14.2 | Prometaphase During prometaphase the definitive mitotic spindle is formed and chromosomes are moved by microtubules into the center of the cell A single kinetochore is attached to microtubules form both spindle poles. Kinectochore: a complex of proteins associated with the centromere of a chromosome during cell division to which the + end of microtubles of the spindle poles attach 9.1-36 9.1-37  14.2 | Prometaphase Chromatids are in a “tug-of-war” between two equally strong centrosomes to generate the metaphase plate Microtubules shorten on one pole and grow on the other pole Plus end can add (polymerize) or lose (depolymerize) subunits Polymerization AND depolymerization at the plus end Depolymerization at the - end 9.1-38  14.2 | Metaphase A cell is in metaphase when the fully condensed chromosomes are all aligned at the metaphase plate (a plane equidistant between the two poles of the spindle) Malignant cell during metaphase Now the cells needs to separate the sister chromatids = anaphase 9.1-39  14.2 | Three types of microtubules The mitotic spindle contains three classes of microtubules Kinetochore microtubules: are connected to chromosomes (after first “finding them via a kinetochore on the chromosome) Astral microtubules: project toward the cell cortex and interact with it thereby orienting the spindle of division Polar microtubules: interact with microtubules from the opposite pole of the cell 9.1-40 14.2 | Anaphase: The Events of Anaphase – microtubule dynamics Tubulin subunits are lost from both ends of kinetochore microtubules Tubulin subunits are lost from the minus ends of polar microtubules Tubulin subunits are added to polar microtubules at the + end Note the role of motor proteins in pushing the polar microtubules apart Pushing apart of the polar microtubules by a four headed kinesin family motor protein 9.1-41 14.2 | Telophase and Cytokinesis Telophase is the final stage of mitosis, the daughter cells return to interphase The mitotic spindle disassembles Nuclear envelopes of the two nuclei are reassembled Chromosomes become dispersed The cytoplasm is partitioned into two cells in a process called cytokinesis 9.1-42  14.2 | Telophase and Cytokinesis Cytokinesis Signals emanating from the central part of the spindle, the spindle midzone, are important for completing cytokinesis Cleavage depends on a belt-like bundle of actin microfilaments (the contractile ring) that form just below the plasma membrane in early anaphase As cleavage progresses, the ring tightens around the cytoplasm Contraction of the ring is generated by interactions between actin and the motor protein, myosin Members of Rho-GTP binding proteins regulate assembly and activation of the contractile ring 9.1-43 Regulation through the cell cycle is controlled by cyclin dependent kinases 9.1-44 Learning objectives: Understand the basics of microtubule structure and dynamics and always be aware of plus v. minus ends Understand what constitutes a microtubule organizing center and the role of a microtubule organizing center Know the functions and directionality of Kinesin and Dynein Know on a basic level what the stages of the cell cycle are (including Go) and what constitutes interphase Know what cytokinesis is For Mitosis: What is a centrosome What is a centriole Understand nucleation of microtubules by gamma TURCs Be clear on the role of microtubule dynamics in all phases of mitosis Be clear on the three types of microtubules and their roles 9.1-45