Lecture 15 MT

Overview of the Cytoskeleton

  • The cytoskeleton consists of three filament systems that can build a wide variety of structures with many functions: microtubules (MTs), intermediate filaments (IFs), and actin filaments.
  • There are numerous accessory proteins that regulate these filaments and expand the possible architectures and functions.
  • Major cellular roles include scaffolding, intracellular transport, force generation for movement and contraction, organization and positioning of organelles, and a key role in cell division machinery.
  • Visual summary (from Figure 9.1): a scaffolding for structure, tracks for movement, force-generating apparatus, framework for organelles, and a component of division machinery.

Major Filament Types: Quick Reference

  • Microtubules (MTs)
    • Subunits: GTP-α-tubulin/β-tubulin (aβ- tubulin heterodimer)
    • Plus end: β-tubulin end; growth preferentially occurs here
    • GTPase activity: yes (tubulin is a GTPase; GTP binding and hydrolysis regulate dynamics)
    • Motor proteins: kinesins and dyneins
    • Major associated proteins: MAPs
    • Polarity: MTs are polar (plus and minus ends)
    • Structure: stiff, hollow tube; outer diameter ≈ 25 nm
    • Distribution: all eukaryotes; cytoplasm
    • Primary functions: support, intracellular transport, cell organization
  • Intermediate filaments (IFs)
    • Subunits: ~70 different proteins, likely incorporated as tetramers
    • Internal organization; no ATP/GTPase activity
    • Major associated proteins: plakins (linkers with other cytoskeletal elements)
    • Structure: tough, flexible, extensible filament; diameter ≈ 10–12 nm
    • Distribution: animals; cytoplasm + nucleus
    • Primary functions: structural support, mechanical strength
  • Actin filaments
    • Subunits: ATP-actin monomers
    • Plus end: barbed (+) end; growth occurs here
    • GTPase/ATPase: ATPase activity
    • Motor proteins: myosins
    • Major associated proteins: actin-binding proteins
    • Structure: flexible, helical filament; diameter ≈ 8 nm
    • Distribution: all eukaryotes; cytoplasm
    • Primary functions: motility, contractility, intracellular transport

Microtubules: Structure and Key Features

  • MTs are the largest cytoskeletal components and come in two main types:
    1) Cytoplasmic microtubules – pervade the cytosol and perform diverse roles such as maintaining axon structure, forming mitotic/meiotic spindles, positioning/transporting vesicles, maintaining cell shape and internal organization.
    2) Axonemal microtubules – organized, stable MTs found in cilia and flagella; include basal bodies from which cilia/flagella attach; the central shaft (axoneme) is a highly ordered MT bundle.
  • MT architecture:
    • Most MTs are straight, hollow cylinders assembled from protofilaments; MTs are typically built from 13 protofilaments.
    • Protofilaments are linear polymers of tubulin heterodimers (ab-heterodimers).
    • The basic subunit dimer consists of α-tubulin and β-tubulin; each subunit has a GTP binding site. In the dimer, the β-subunit binds GTP or GDP and is the site of hydrolysis during MT dynamics; α-tubulin binds GTP that is trapped and not hydrolyzed under normal conditions.
    • All protofilaments within a MT are oriented in the same direction (defining a distinct plus end and minus end).
  • Dimers and polarity:
    • Dimers form into protofilaments; MTs have a distinct polarity with a plus end and a minus end.
    • The plus end is where most growth occurs; the minus end is often anchored, typically at the centrosome in vivo.
  • MT dimensions and distribution:
    • Outer diameter ≈ 25 nm25\ \text{nm}
    • MTs are found in all eukaryotes, with cytoplasmic MTs predominant in the cytoplasm.

Tubulin: Subunits, GTP, and Dynamics

  • The basic MT building block is the tubulin heterodimer (α-tubulin + β-tubulin).
  • GTP binding and hydrolysis:
    • Each monomer has a GTP-binding site.
    • The GTP bound to α-tubulin is trapped and not hydrolyzed; β-tubulin can bind GTP or GDP, and hydrolysis occurs after incorporation into the MT.
    • Tubulin is a GTPase; GTP promotes dimer interactions and incorporation into MTs, but hydrolysis is not required for initial assembly (it occurs after incorporation).
    • Growing MTs have GTP-bound β-tubulin at the plus ends; shrinking MTs have GDP-bound β-tubulin at the ends.
    • The presence of a GTP cap at the plus end stabilizes the MT and prevents rapid depolymerization.
  • Dynamic states:
    • GTP-tubulin at the plus end stabilizes filament; hydrolysis to GDP within the lattice destabilizes and promotes depolymerization when the cap is lost.
    • The GTP cap prevents subunit removal and maintains growth; loss of the cap leads to rapid shrinkage (catastrophe).
  • Key end-state differences:
    • Plus end: dynamic and growth-prone; GDP destabilizes the end if hydrolysis lags behind addition.
    • Minus end: often anchored (especially at centrosomes), dynamics are typically limited there in vivo.

Microtubule Assembly and Kinetics

  • In vitro MT assembly is influenced by tubulin dimer concentration.
  • Phases of MT polymerization in vitro:
    • Nucleation: dimers aggregate into oligomers that act as nuclei for MT growth; this lag phase is due to slow initial nucleation.
    • Elongation: rapid addition of tubulin dimers at MT ends; overall MT length increases more quickly than nucleation.
    • Plateau: when free tubulin becomes limiting, assembly balances disassembly.
  • Critical concentration (Cc):
    • The tubulin concentration at which MT assembly is exactly balanced by disassembly.
    • If [tubulin] > Cc, MTs grow; if [tubulin] < Cc, MTs shrink.
    • In equation form (conceptual): if rgrowth = kon[T] − koff, then at [T] = Cc, rgrowth = 0.
  • End-specific growth rates:
    • The plus end polymerizes more rapidly than the minus end in vitro.
    • In vivo, the minus end is usually anchored at the centrosome, confining most dynamic activity to the plus end.
  • In vitro vs in vivo dynamics:
    • In vitro, MTs can grow or shrink at ends; in vivo, regulatory factors and cellular context constrain dynamics, particularly at the minus end.

Dynamic Instability of Microtubules

  • Dynamic instability describes rapid transitions between growing and shrinking states at MT plus ends.
  • Conceptual model: one population of MTs grows by polymerization at the plus ends, while another population shrinks by depolymerization.
  • Mechanistic steps (typical sequence):
    1) The growing tip contains GTP-bound β-tubulin subunits.
    2) GTP is hydrolyzed as the protofilament extends.
    3) GDP-bound β-tubulin accumulates at the end after hydrolysis, destabilizing the end.
    4) The presence of GDP-tubulin at the end causes conformational strain and curling of protofilaments, leading to rapid depolymerization (catastrophe).
  • Key consequence:
    • Growing MTs have a GTP cap at the plus end; shrinking MTs expose GDP-tubulin ends.
    • The dynamic cycle is driven by GTP hydrolysis in β-tubulin and the interplay between assembly and disassembly rates.
  • Important nuance:
    • The GTP bound to α-tubulin is never hydrolyzed; hydrolysis occurs on the β-tubulin after incorporation into the MT lattice.

GTP Hydrolysis and the “GTP Cap” Concept

  • All MT subunits in a lattice can carry GTP initially, but hydrolysis occurs after incorporation into the MT lattice, predominantly influencing the stability of the growing end.
  • Distinctions:
    • GTP-bound β-tubulin at the plus end supports continued polymerization.
    • GDP-bound β-tubulin at the end weakens lateral and longitudinal contacts, leading to catastrophe.
  • Diagrams and textual notes (as described in Fig. 9.21):
    • 1) The growing tip contains GTP-bound β-tubulin subunits.
    • 2) Hydrolysis of GTP as the tube extends.
    • 3) GDP-bound β-tubulin at the end.
    • 4) GDP-induced conformational strain promotes curling and rapid depolymerization.
  • Practical takeaway:
    • The balance of GTP hydrolysis and subunit addition controls MT length and stability in cells.

Drug Interventions Targeting Microtubules

  • Colchicine (from Colchicum autumnale): binds tubulin and prevents polymerization, leading to depolymerization; clinical use: treatment of gout (reduces inflammation by blocking leukocyte migration).
  • Paclitaxel (Taxol) from Pacific yew tree: stabilizes microtubules by binding β-tubulin, preventing depolymerization; clinical use: chemotherapy for ovarian, breast, and lung cancers.
  • Nocodazole: synthetic; binds tubulin and inhibits polymerization; primarily a research tool to synchronize cells in mitosis.
  • Vinblastine: from Madagascar periwinkle; binds tubulin dimers and prevents MT assembly; chemotherapy for Hodgkin’s lymphoma and testicular cancer.
  • Practical implication: These compounds are widely used to study MT dynamics and are clinically important in cancer therapy, as well as tools to manipulate cell division and motility.

Microtubule Organizing Centers (MTOCs)

  • Role: MTs originate from MTOCs, which nucleate MT assembly and anchor the minus ends.
  • In interphase animal cells, the primary MTOC is the centrosome near the nucleus.
  • Centrosome composition:
    • Two centrioles oriented at right angles to each other.
    • Surrounding pericentriolar material (PCM) with accessory proteins that nucleate MTs.
    • Centrioles participate in basal body formation for cilia/flagella.
    • Cells missing centrioles can still divide, but with poorly organized mitotic spindles (not all organisms depend on centrioles to divide).
  • Centriole structure:
    • Centrioles are composed of nine sets of triplet MTs.

Gamma-Tubulin Ring Complexes (γ-TuRC) and Nucleation

  • MT nucleation is accelerated by γ-tubulin ring complexes (γ-TuRCs) located in the PCM of the centrosome.
  • Structure and function:
    • γ-TuRCs contain γ-tubulin subunits and associated proteins (GRiPs).
    • They nucleate MTs away from the centrosome, providing the initial template for MT growth.
    • There are 13 γ-tubulin subunits per turn in the γ-TuRC, forming a ring that templates MT minus ends and helps define MT polarity.
  • Consequences:
    • Loss of γ-TuRC function prevents MT nucleation.
    • γ-TuRCs contribute to establishing MT polarity and minus-end capping at the nucleation site.
  • Non-centrosomal nucleation:
    • MT nucleation can also occur at non-centrosomal sites that recruit γ-TuRCs, enabling MT growth away from the centrosome in specialized contexts.

Centrosome, MTOC Polarity, and MT Organization in Dividing Cells

  • MTs extend outward from the MTOC toward the cell periphery, with minus ends anchored at the MTOC and plus ends growing outward.
  • In non-dividing cells, MT orientation can vary depending on cell type and function.
  • The MTOC influences the number of MTs in a cell; e.g., high MT-nucleating activity is observed during prophase/metaphase when the spindle forms.

Microtubule-Binding Proteins (MAPs) and Regulation of MT Stability

  • MT stability in cells is controlled by a diverse set of MAPs, grouped functionally as:
    1) ATP-driven MAPs that power vesicle/organelle transport or generate sliding forces between MTs (often motor proteins).
    2) MT-stabilizing and bundling proteins.
    3) Plus-End Tracking Proteins (+-TIPs) that associate with growing MT plus ends.
    4) MT-destabilizing and severing proteins that promote disassembly.
  • Representative MAPs and roles:
    • Tau: stabilizes MTs and promotes tight bundling in axons; MAP2 promotes looser bundles in dendrites.
    • Stathmin/Op18: binds tubulin heterodimers and prevents polymerization.
    • Catastrophins (kinesins): promote catastrophe by enhancing end peeling and disassembly at MT ends.
    • Katanin: a MT-severing protein that breaks MTs to regulate network remodeling.
    • EB1 (a +TIP): associates with GTP-tubulin at growing ends to stabilize MTs and promote plus-end tracking; also important at kinetochores during mitosis.
  • A MAP may interact at regular intervals along the MT wall, increasing stability or density of MTs and coordinating interactions with other motor or MAP proteins.
  • Illustrative notes:
    • The Tau–MAP2 distinction demonstrates how MAPs can influence MT organization in different neuronal compartments (axons vs. dendrites).

Polarity, Nucleotides, and MT Dynamics: Key Concepts to Master

  • MT roles and architecture:
    • Roles of the cytoskeleton in cells include scaffolding, tracks for transport, force generation, spatial organization of organelles, and involvement in cell division.
    • Subunits that comprise MTs include the α/β-tubulin heterodimer with distinct nucleotide states that govern dynamics.
    • Protofilaments confer MT polarity; plus and minus ends differ chemically and in dynamics.
  • Dynamics and regulation:
    • Dynamic instability underpins MT remodeling required for cell division and adaptive cellular architecture.
    • Nucleotides (GTP on β-tubulin and non-hydrolyzable GTP on α-tubulin) govern MT assembly and stability.
    • MTOCs (centrosomes) and γ-TuRCs play central roles in nucleation and minus-end anchoring, shaping MT networks.
  • Experimental and clinical relevance:
    • Understanding MT dynamics is central to cancer biology due to mitotic spindle function and the mechanism of action of MT-targeting drugs.
    • Drugs that stabilize or destabilize MTs alter cell division and can be used therapeutically or as research tools.

Summary: Core Takeaways to Remember

  • MTs are polar, 13-protofilament hollow tubes of diameter ≈ 25 nm25\ \text{nm}, built from GTP- or GDP-bound tubulin dimers; plus end grows faster and is β-tubulin exposed; minus end is often anchored at the MTOC.
  • Tubulin dimers add to MT ends in a process that requires GTP and Mg²⁺; hydrolysis occurs after incorporation, creating a GDP-tubulin lattice that can destabilize the MT if a GTP cap is lost.
  • Dynamic instability is the rapid transition between growth and shrinkage, driven by GTP hydrolysis and end conformation; growth ends carry a GTP cap that stabilizes growth.
  • The centrosome/MTOC, via γ-TuRCs, nucleates MTs and anchors the minus ends; γ-TuRCs contain 13 γ-tubulin subunits per turn and cap the minus end, guiding MT polarity.
  • MTs are regulated by a diverse family of MAPs, which can stabilize, destabilize, bundle, or track MT plus ends; Tau and MAP2 illustrate compartment-specific MT organization in neurons.
  • MT-targeting drugs (e.g., Colchicine, Taxol, Nocodazole, Vinblastine) illustrate how manipulation of MT dynamics can influence cell division and therapy.

End-of-Section Objectives (What you should be able to explain)

  • Describe the roles of the cytoskeleton and the major functions of MTs, IFs, and actin filaments.
  • Identify the subunits that comprise MTs, protofilament structure, and MT polarity (plus end vs minus end).
  • Explain dynamic instability and how GTP hydrolysis in tubulin regulates MT growth and shrinkage.
  • Define the critical concentration Cc for MT assembly and describe how MT growth depends on [tubulin].
  • Distinguish between cytoplasmic MTs and axonemal MTs and explain their respective roles.
  • Explain the concept and structure of the MTOC, centrosome, centrioles, and PCM, and how they organize MTs.
  • Describe γ-TuRCs, their structure (13 γ-tubulin subunits per turn), and their role in nucleating MTs away from the centrosome.
  • Understand the different classes of MAPs and give examples of how they regulate MT stability and dynamics (e.g., Tau, MAP2, EB1, Stathmin, Katanin).
  • Be able to relate these concepts to real-world relevance, such as MT roles in cell division, transport, and disease