Welcome to the Wonderful World of Actin and Actin Binding Proteins
key words: cytoskeleton; actin; actin filaments = microfilaments; actin-binding proteins; spectrin; alpha-actinin
The Cytoskeleton.
One distinguishing feature that separates the eukaryotes from prokaryotes is the presence of the cytoskeleton in the former, and apparent absence of it in the latter. This suggests that the cytoskeleton was an important factor in eukaryote evolution. The function of the cytoskeleton is to maintain cellular shape, and is involved in intracellular organization, cell polarity, cell adhesion, and in some cases, motility.
The cytoskeleton is composed of several different protein components, which I will describe in some detail below. In general, cytoskeletal proteins polymerize to form long chains, giving the appearance of strand-like or fibrous structures. There are three general classes of cytoskeletal fibers: (1) microtubules, (2) intermediate filaments, and (3) actin filaments.
Compared to the other cytoskeletal fibers, the microtubule is rather large (~15 to 35 nm diameter). Microtubules are composed of a globular protein, tubulin. The tubulin subunit is a heterodimer of alpha- and beta-tubulin (Fig-1a). The microtubule itself is made up of 13 "proto-filaments", which are each composed of alternating alpha- and beta- subunits (Fig-1b). These protofilaments are cylindrically arranged to form a hollow tube -- it is this arrangement of proto-filaments that makes up the microtubule (Fig-1c). Microtubules are polar molecules -- i.e. they have a fast growing "plus end" and a slow growing "minus end". These strands are in a constant state of flux, termed "dynamic instability" (i.e. they continually grow and fall apart). Free GTP (guanosine triphosphate) binds to the beta-tubulin, which alters its protein structure and enables it to join to the growing end of the strand. Then, there is a delayed GTP hydrolysis reaction to yield GDP (guanosine diphosphate). Thus, at the terminus of the filament a GTP cap is present (i.e. tubulin subunits are bound to GTP), while further down the strand, tubulin subunits are bound to GDP. The presence of GDP creates a weak bond between tubulin subunits, and therefore the filament is more likely to de-polymerize. In other words, if all the GTP is hydrolysed in the filament, the filament is susceptible to falling apart (i.e. under a microscope, the microtubules are observed to shrink or even disappear). There are various proteins that are able to stablize the microtubule, preventing depolymerization -- these are known as "cap-proteins" or MAPs (Microtubule-Associated Proteins). There are also various other chemicals that can either stabilize or destabilize microtubules (and these chemicals are often used in research). For example, colchicine binds to free tubulin subunits and prevents polymerization (e.g. colchicine is often used stop cell division at the metaphase stage, since spindle fibers are made of microtubules). Another chemical is taxol, which binds to the microtubules and stabilizes them.
FIG-1: A) Tubulin subunits alpha and beta, B) Protofilament composed of tubulin subunits, C) Microtubule. (dotted lines below diagrams indicate continuation of the structure).
Eukaryotic cells may have a structure that attaches to one end of the microtubule (e.g. to the "minus" end) -- these are MTOCs (Microtubule-Organizing Centers). One example of an MTOC is the centrosome (Fig-2a, arrow). The centrosome is a structure located near the nucleus of a cell and is composed of two centrioles arranged perpendicularly to one another (Fig-2b). Centrioles are made of microtubules arranged as "9 and 0" triplets. The centrosome is thought to play some role during cell division (i.e. in mitosis), however, the details of how it does this are not certain. The centrosome is present in most animal cells, but absent in plant cells (which suggests that centrosomes are not required for cell division -- at least in plants). Rather, plant cell MTOCs are referred to as "poorly defined regions". Thus, there are morphological differences of MTOCs within the eukaryotes, yet the mechanism may be conserved, since they use tubulin proteins and organize microtubules.
FIG-2: A) Cell with nucleus (N) and centromere (arrow). B) Centromere (with perpendicular centrioles) as it would look under a microscope or in an electron-micrograph.
Another group of cytoskeletal proteins are the intermediate filaments (IFs). IFs have an intermediate size between microtubules and actin filaments (7 to 10 nm diameter). I will not go into detail about intermediate filaments, since the group is composed of numerous and different proteins (e.g. vimentin, lamin, keratins), and my focus here (below) is really on the actin-based cytoskeleton. There are, however, two general types of IFs: (1) cytoplasmic IFs, and (2) nuclear lamins. Cytoplasmic IFs are for mechanical stress and cell-to-cell junctions. Nuclear lamins create a meshwork beneath the inner nuclear membrane. One important difference to note between IF proteins and proteins for microtubules or for microfilaments, is that most IF protein subunits are filamentous, rather than globular (i.e. tubulin and actin subunits are globular).
Actin filaments (or "microfilaments" -- these terms are used interchangeably) are the smallest of the cytoskeletal fibers (3 to 6 nm diameter). Microfilaments are flexible doubled-stranded fibers composed of polymers of the protein actin (contrast this structure to microtubules, which are hollow tubes composed of 13 protofilaments). Actin is present in all eukaryotes, and microfilaments are typically found in the cell cortex (i.e. just beneath the cell membrane). The actin subunit is globular and has a molecular mass of 43 kDa. As with microtubules, actin filaments are also dynamic and polar molecules (i.e. they have a fast growing "plus end" and a slow growing "minus end"). Free actin binds ATP (adenosine-triphosphate, as opposed to GTP in tubulin subunits) which enables polymerization, while ATP hydrolysis to ADP favours depolymerization. Unlike microtubules which undergo dynamic instability, actin filaments may use a different dynamic process. "Treadmilling" occurs when there is no net change in the length of the filament -- i.e. actin subunits are continually added to the "plus end" and lost from the "minus end". It could be that actin filaments undergo both processes under different situations. By way of terminology, as used in the literature, actin is referred as "F-actin" if it is in the polymeric "filamentous" form, and "G-actin" if it is in the monomeric "globular" form. As with MTs, there are various chemicals that can bind to actin monomers or filaments to prevent or promote polymerization, enhance microfilament stability, or to favour depolymerization. For example, cytochalasin and phalloidin are two commonly used chemicals in research dealing with the actin cytoskeleton. Cytochalasin prevents actin polymerization by binding to the "plus end" of the filament. Conversely, phalloidin binds to the sides of the actin filament and prevents depolymerization. Both cytochalasins and phalloidins are isolated from different fungal species, and are made commercially available for research. This is a good example of why some fungi (e.g. mushrooms, such as Amanita) are toxic to humans (i.e. these toxins interfere with the proper functioning of the cytoskeleton). Another type toxin that has come into research use, which also inhibits actin polymerization, is latrunculin. Latrunculins are isolated from a species of red sea sponge (Latrunculin magnifica). Presumably, these toxic chemicals in fungi and sponges evolved as defense mechanisms against animal predators.
Actin Binding Proteins.
The actin-based cytoskeleton functions for bearing of tension and for compression resistance (i.e. it is like a shock suspension system, which gives mechanical strength and maintains structural integrity of a cell). In fact, there are different microfilament arrangements for a variety of functional purposes. There are, however, two general classes of microfilament arrangement: (1) bundles (parallel and contractile), and (2) gel-like networks. Different proteins (i.e. actin-binding proteins) mediate the different arrangements. Parallel bundles are structures where microfilaments are oriented with the same polarity (i.e. plus ends are all "pointed" in the same direction) and which are closely spaced. In this case, fimbrin (a 68 kDa protein which works as a monomer) is the protein link spanning 14 nm between microfilaments. Contractile bundles are more loosely spaced arrangements of microfilaments which are oriented with opposite polarities. These are linked by alpha-actinin, a flexible 100 kDa protein which functions as a homodimer, spacing the actin filaments by 40 nm. Lastly, there are gel-like networks of microfilaments which may be linked by any of a variety of actin-binding proteins. One example is that microfilaments can be linked by filamin -- a 270 kDa, V-shaped, dimeric protein.
Below, I will describe some background information on the particular group of proteins that I work on. Then I will go into some detail about my own undergraduate research into this area.
The Spectrin-Superfamily
The spectrin superfamily is comprised of spectrin, alpha-actinin, and dystrophin (Lorenz et al., 1995), and the spectrin-based membrane skeleton is considered to be ubiquitous among eukaryotes (Thomas et al., 1997). The spectrin superfamily is a group of actin binding proteins that are considered to be related through an alpha-actinin-like ancestral protein dating back at least 500 million years (Muse et al. 1997). Spectrins have been identified in a number of diverse organisms, such as Acanthamoeba, Paramecium, Chlamydomonas (Lorenz et al. 1995), a variety of plant species, such as rice (Faraday and Spanswick, 1993), maize, carrot, and potato (de Ruijter and Emons, 1993), as well as chicken, human, and a few other species. The group is characterized by evolutionarily conserved N-terminal actin-binding domains, and homologous tandem repeats of ~106 amino acid long segments.
The reasons for studying this group of proteins is not merely for academic interest (e.g. with respect to evolution -- although, for me, it is). There are also medically relevant implications. In particular, deficiency in spectrin (due to genetic abnormalities) can lead to anemia. In fact, a model system for studying spectrin is the human erythrocyte (i.e. red blood cell) cytoskeleton (Speicher and Marchesi, 1984). The concave shape of human red blood cells (RBCs) is maintained by spectrin and actin, which are the predominant proteins in the RBC cytoskeleton. As RBCs circulate in the blood system, they are subject to collisions with the walls of arteries, veins, and capillaries, and therefore must have a membrane structure with elastic properties. It is this spectrin-based cytoskeleton that provides the compressible meshwork for stress resistance. Another application could deal with agriculture and/or industry. Tip growth is a characteristic property of fungi, pollen tubes, and root hairs (Heath, 1995). Understanding fungal growth would help in controlling pathogenic or "problematic" fungi (such as molds), or industrially useful species (such as Saccharomyces). Spectrin is considered to be a membrane associated protein (Kaminskyj and Heath, 1995), therefore somehow plays a role during tip growth in these model systems. Thus, understanding how these proteins are localized into membrane cytoskeletons, as well as characterizing their interactions with other cellular components, has wide reaching applications.
alpha-Actinin and Spectrin
The two related proteins, alpha-actinin and spectrin, have several features in common, but there are also some differences. Both are structural proteins (as opposed to enzymatic proteins) and they bind to actin filaments.
On the other hand, alpha-actinin is a homodimer, whereas spectrin is a heterotetramer. The alpha-actinin subunit is also considerably smaller than the spectrin subunits. It is thought that spectrin evolved from an alpha-actinin-like ancestral protein. I will now describe some of the features that are known about these proteins.
The alpha-actinin subunit is a 100 kDa filamentous protein, and functions as a homodimer (i.e. two identical subunits combine to form a structural protein which is able to cross-link actin filaments). The subunits are oriented in antiparallel fashion and dimerize primarily through electrostatic interactions, but also by van der Waals forces, hydrogen bonding, and hydrophobic interactions (Viel, 1999).
The alpha-actinin subunit essentially has three domains: an N-terminal actin-binding domain, a rod domain made of four tandem repeats (R1, R2, R3, R4) and linker regions, and a C-terminal Calmodulin-like (calcium-binding) domain.
FIG-3: Schematic of alpha-actinin. [ref: from fig-1 in Veil (1999) FEBS letters. 460:391-394]
Human erythrocyte spectrin is a heterotetramer composed of alpha- and beta- spectrin subunits. The alpha- and beta-subunits dimerize in antiparallel fashion, and then two dimers unite head-to-head to form the tetramer. The spectrin subunits are larger and slightly more complex than the alpha-actinin subunit (Thomas et al. 1997). The alpha-subunit is 220 kDa, and has 23 segments in total arranged as follows: #0 = partial repeat, #1-9 and #11-21 = 20 spectrin repeats, #10 = SH3 domain embedded in #9, and #22 = a Calcium-binding domain. The beta-subunit is 240 kDa, and has 19 segments: #1 = ABP at the N-terminus, #2-17 = 16 spectrin repeats, #18 = alpha-/beta- head to head interactions, and #19 = C-terminal pleckstrin homology (PH) domain. A much heavier beta-chain (BH-) has been found in Drosophila and C. elegans (Thomas et al., 1997). This heavy beta-chain is 470 kDa, and has 33 segments: #1 = ABD at the N-terminus, #2-6 and #8-31 = 29 repeats, #7 = SH3 domain, #32 =partial repeat, #33 = C-terminal PH domain. The critical features to note are that the alpha-subunit lacks the ABD and has 20 repeats, while the beta-subunit has an ABD at its N-terminus. Also note that segments in the heavy and lighter beta-chains are arranged similarly.
Fig-4A: A model of the Spectrin heterotetramer. [ref: fig-4 from Speicher and Marchesi (1984) Nature. 3121:177-180]
Fig-4B: A simplified schematic of the triple-helices in spectrin. [ref: fig-3 from Speicher and marchesi (1984) Nature. 3121:177-180]
Fig-4C: Schematic of the Spectrin heterotetramer. [ref: from fig-1 in Veil (1999) FEBS letters. 460:391-394]
A key feature of spectrin and alpha-actinin subunits are the repeats. Each repeat is composed of about 106 amino acids which form a triple helix structure plus a helical linker region. Two papers published in the same issue of the journal Cell provided confirmation of the structure and the flexible nature of these repeats (Grum et al., 1999; Djinovic-Carugo et al., 1999). I will not discuss the results from these studies, but if you are interested in the topic (and especially, if you are interested in structural biology) you should read those references. At some point, I may decide to go into more detail about the evolution of the spectrin superfamily (however, for one interesting explanation, see Veil, 1999).
References:
- Djinovic-Carugo, K., Young, P., Gautel, M. and Saraste, M. (1999) Structure of the alpha-actinin rod: molecular basis for cross-linking of actin filaments. Cell. 98: 537-546
- Faraday, C.D. and Spanswick, R. (1993) Evidence for a membrane skeleton in higher plants: a spectrin-like polypeptide co-isolates with rice root plasma mebranes. FEBS letters. 318:313-316
- Grum, V.L., Li, D., MacDonald, R.I. and Mondragon, A. (1999) Structures of spectrin suggest models of flexibility. Cell. 98:523-535
- Heath, I.B. (1995) Integration and regulation of hyphal tip growth. Canadian Journal of Botany. 73: S131-S139
- Kaminskyj, S.G.W. and Heath, I.B. (1995) Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth. Journal of Cell Science. 108: 849-856
- Lorenz, M., Bisikirska, B., Hanus-Lorenz, B., Strzalka, K. and Sikorski, A.F. (1995) Proteins reacting with anti-spectrin antibodies are present in Chlamydomonas cells. Cell Biology International. 19: 625-632
- Muse, S.V., Clark, A.G., and Thomas, G.H. (1997) Comparisons of the nucleotide substitution process among repetitive segments of the alpha- and beta-spectrin genes. Journal of Molecular Evolution. 44:492-500
- de Ruijter, N. and Emons, A. (1993) Immunodetection of spectrin antigens in plant cells. Cell Biology International. 17: 169-182
- Southern, E.M> (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology. 98: 503-517
- Speicher, D.W. and Marchesi, V.T. (1984) Erythrocyte spectrin is comprised of many homologous triple helical segments. Nature. 311: 177-180
- Thomas, G.H., Newbern, E.C., Korte, C.C., Bales, M.A., Muse, S.V., Clark, A.G. and Kiehart, D.P. (1997) Intragenic duplication and diverghence in the spectrin superfamily of proteins. Molecular Biology and Evolution. 14:1285-1295
- Viel, A. (1999) alpha-Actinin and spectrin structures: an unfolding family story. FEBS letters. 460: 391-394
|
Get your own Free Home Page