5.12 Cytoskeletal systems
In the descriptions above we have often used words implying delivery of a cargo contained in a vesicle to a specific target site. The vesicles do not slosh around in a cytoplasmic soup, rather they are conveyed to their destinations by the cytoskeletal system.
The cytoskeleton is a characteristic feature of eukaryotic cells; homologues to all the major proteins of the eukaryotic cytoskeleton can be found in prokaryotes, although the sequence comparisons indicate very distant evolutionary relationships. As the name ‘cytoskeleton’ implies it is generally presented as the structure that maintains cell shape and permits motion, but this is only fully applicable to animal cells; in plants and fungi the cell wall largely determines cell shape and motion is limited (though in fungi the invasive hyphal tip could be viewed as being a motile apex). The cytoskeletal functions that apply throughout the eukaryotes are to provide for intracellular transport of vesicles and organelles, and segregation of chromosomes during nuclear division.
There are three main components making up the eukaryotic cytoskeletal system: actin filaments or microfilaments, intermediate filaments, and microtubules:
- actin filaments/microfilaments are solid rods about 7 nm in diameter made of two chains of the globular protein called actin (one of the most abundant proteins in nature). These filaments are contractile and contribute to shape changes, cell-to-cell or cell-to-wall connections, signal transduction, cytokinesis, and cytoplasmic streaming;
- intermediate filaments are a very broad class of fibrous proteins forming structural fibres in the range 8 to 12 nm diameter. They also participate in cell-to-cell and cell-external connections, but mostly function as tension-bearing elements maintaining shape and rigidity of the animal cell, and the structure of membranous structures like the nuclear envelope. The first intermediate filament gene to be characterised in filamentous fungi, the A. nidulans mbmB gene product, co-localises with mitochondria, and deletion of mdmB affects mitochondrial morphology and distribution;
- microtubules are straight, hollow cylinders about 25 nm in diameter, usually composed of 13 protofilaments, which are polymers of α- and β-tubulin. They have a very dynamic behaviour, and carry out a variety of functions, ranging from transport to structural support.
The importance of actin in budding yeast is evident from the phenotype changes resulting from mutation of the gene that encodes actin, which include:
- gross morphological defects,
- abnormal chitin deposition,
- defective bud site selection,
- abnormal nuclear segregation,
- abnormal cytokinesis,
- abnormal distribution of intracellular organelles,
- abnormal secretion and uptake,
- altered sensitivities to the environment (temperature, osmotic and ion concentrations).
The actin protein has a molecular weight of about 42 kDa (375 amino acids), which exists as a monomer (referred to as globular or G-actin) or as a linear polymer of the monomer, known as filamentous or F-actin. It is the filaments that are so important in morphogenesis, organelle movements and cytokinesis; the microfilaments are extremely dynamic structures that can be rapidly modified by interactions with a number of actin binding proteins (ABPs)(Sutherland & Witke, 1999; Walker & Garrill, 2006). The actin cytoskeleton of filamentous fungi is required for polarity establishment and maintenance at hyphal tips and for formation of a contractile ring at sites of septation. Actin-related proteins, known as formins, have been identified as independent nucleators of actin polymerisation. Filamentous fungi contain a single formin that localises to both hyphal tips and the sites of septation (Xiang & Plamann, 2003).
Microtubules and their associated proteins are involved with as wide a range of intracellular functions as are actin microfilaments (indeed, the two work together in most instances), particularly transport and positioning of organelles, vesicles and nuclei. Microtubules are made up of dimers of α- and β-tubulin that assemble into cylindrical tubes with a diameter of about 25 nm but varying greatly in length. The polymerisation dynamics of microtubules are central to their biological functions. Even in vitro, purified tubulin dimers continuously self-assemble and disassemble; microtubules polymerise and elongate until they randomly switch to depolymerisation (the switch is termed catastrophe). Depolymerisation leads to rapid shortening of the microtubule, resulting either in their complete disappearance or in a switch back to elongation (which is termed rescue). This dynamic instability is an essential property of microtubules as it enables them to perform mechanical work. An elongating microtubule is polarised, having a rapidly elongating plus end, and a slowly or non-elongating minus end (in cells, the minus end is usually the anchor point).
In vivo, microtubules utilise the energy of GTP hydrolysis to drive their dynamic instability (= polymerisation/depolymerisation). Microtubules are formed by assembly of α- and β-tubulin dimers. The first stage of their formation is called nucleation, which requires Mg2+ and GTP. During nucleation, α- and β-tubulins join to form a dimer. Each dimer carries two GTP molecules, but it’s the one on the β-tubulin that is hydrolysed to GDP when a tubulin molecule adds to the microtubule. Dimers attach to other dimers to form oligomers, 13 at a time, forming rings 25 nm in diameter. The longitudinal rows of dimers are called protofilaments. Nucleation is relatively slow, but once the microtubule is fully formed the second phase, called elongation, proceeds more rapidly. The length of microtubules is determined by elongation rate, shortening rate, catastrophe frequency and rescue frequency. The cell controls its microtubule network by modifying these parameters, and this in turn affects the intracellular transport processes.
A wide range of microtubule-binding proteins is involved in these organising and maintenance processes. Some microtubule-binding proteins specifically associate with the plus ends of growing microtubules. These proteins are called plus end tracking proteins (or +TIPs). They form clusters at the ends of growing microtubules and regulate microtubule dynamics. A specific animal example is CLIP-170 which is a ‘Cytoplasmic Linker Protein’ that binds to the growing plus end of microtubules, enhances microtubule assembly, and is involved in interactions between endosomal membranes and microtubules. CLIP-170 homologues in Saccharomyces cerevisiae stabilise microtubules by reducing all four parameters of dynamic instability, while they suppress catastrophes in Schizosaccharomyces pombe (remember, these two yeasts are related, but belong to different phylogenetic lineages [CLICK HERE to see it now]. CLIP-170 homologues of Aspergillus nidulans promote microtubule growth by doubling the rescue frequency. Evidently, organism, tissue and cell type specific functions can be expected in these and other microtubule associated proteins (MAPs). MAPs include the microtubule motors, kinesin and dynein, which ‘walk’ along the microtubules (Fig. 7, below) to provide the characteristic motility. They work in opposite directions, kinesins move their cargo towards the plus end, dynein walks towards the minus end. Dynein motors are concentrated at microtubule plus ends in fungi, where they influence catastrophe and rescue rates in S. cerevisiae, A. nidulans and Ustilago maydis, thus functioning like +TIPs as well as being minus-end-directed motors.
It may be worth mentioning here that the antifungal drug griseofulvin inhibits mitosis in fungi by binding to MAPs and the fact that it is clinically useful suggests that there are major differences between the MAPs of humans and those of fungi.
Updated January 18, 2017