Crossroads on Cytoskeletal Highways

Abstract

As prokaryotes shed their tough, resilient cell walls and evolved into eukaryotes, intricate and dynamic infrastructures emerged to enable cells to survive against the physical and chemical assaults of the environment, and to allow organisms to develop elaborate cell shapes and multicellular dependency. Cytoplasmic microtubules (MTs) evolved for intracellular trafficking of vesicles, organelles, and proteins, and actin microfilaments (MFs) were utilized for cell polarity, contractile, and migratory processes. A third cytoskeletal network, intermediate filaments (IFs), evolved later as exoskeletal armors gave way to endoskeletons and higher eukaryotes acquired the need for mechanical integrators of the cytoplasm. Thus, although many lower eukaryotes survive perfectly well without cytoplasmic IFs, humans lacking IFs can become the unhappy bearers of degenerative disorders. IFs are especially abundant in vertebrate tissues such as epidermis and muscle that undergo substantial physical stress, reflective of their ability to impart intracellular mechanical strength. While cell biologists were elucidating the roles of cytoskeletal networks and the intricacies of proteins associated with each network, they began to observe that agents perturbing one network often affected the others, and that functions ascribed to one network were sometimes also features of another. In the past few years, connector proteins have been discovered that associate with multiple cytoskeletal networks, integrating old functions and creating new ones. As the roles of these connectors unravel, it has become increasingly clear that they are tailored to suit the tissue-specific needs for cytoskeletal dynamics in cells. The functions of these connectors are as intriguing and complex as the networks themselves. The cytoarchitecture of higher eukaryotic cells varies tremendously, ranging from long and slender neurons to resilient, stratified epidermal cells able to survive at the body surface without hair, feathers, or an exoskeleton to protect them. IF networks and their associated proteins contribute heavily to this heterogeneity. While the ability of IFs to act as mechanical reinforcers seems to be their universal property, IFs meet specialized cytoskeletal requirements through their divergent sequences and expression patterns, which in turn lead to versatility in their physical properties and relative abundance, and in the proteins that associate with them. As IFs evolved to provide infrastructures, it became equally advantageous to utilize IFs in specialized ways by integrating them with other cellular components. In stratified epithelia, for instance, IFs attach to desmosomes and participate in cell–cell adhesion, and to hemidesmosomes, specialized integrin-mediated sites of cell–substratum adhesion. These attachments are mediated by plakins, a family of enormous (>200 kDa) coiled-coil dimeric proteins, related by sequence and by their ability to act as molecular bridges between cytoskeleton and other cellular structures (Table 1). The prototypes of this family are desmoplakin, bullous pemphigoid antigen 1e (BPAG1e), and plectin, all able to associate with IFs through their carboxy tail segments. Plakins complete their links between IFs and adhesive junctions through defined interacting domains. For example, plectin and BPAG1e (the epithelial form of BPAG1) each bind to hemidesmosomes (Figure 1A).Table 1Key Cytoskeletal Connectors and Their Biological PropertiesTable 1Key Cytoskeletal Connectors and Their Biological Properties Plakin functions have been examined through gene knockout technology, and in some organs, particularly skin, these connector molecules play structural roles that partially overlap with the functions of IFs to which they connect (1Andra K Nikolic B Stocher M Drenckhahn D Wiche G Genes Dev. 1998; 12: 3442-3451Crossref PubMed Scopus (151) Google Scholar, 3Gallicano I.G Kouklis P Bauer C Yin M Vasioukhin V Degenstein L Fuchs E J. Cell Biol. 1998; 143: 2009-2022Crossref PubMed Scopus (250) Google Scholar). Thus, the loss of BPAG1e severs keratin IFs from hemidesmosomes creating intracellular fragility at the base of the epithelium. No longer withstanding mechanical shear forces, the epithelium ruptures, causing a skin blister. In humans, this condition is known as epidermolysis bullosa simplex (EBS), most frequently caused by IF-disrupting mutations in the keratins themselves (2Fuchs E Cleveland D Science. 1998; 279: 514-519Crossref PubMed Scopus (793) Google Scholar). Humans lacking plectin also display EBS, but in addition show signs of muscular dystrophy, reflective of plectin's dual expression in epidermis and muscle (1Andra K Nikolic B Stocher M Drenckhahn D Wiche G Genes Dev. 1998; 12: 3442-3451Crossref PubMed Scopus (151) Google Scholar and references therein). Thus, as underscored by the physiological importance of BPAG1 and plectin, maintaining IF cytoarchitecture through plakins is likely to be a key feature of many cells. In some cases, the loss of a plakin affects cellular junctions as well as IF architecture. Without desmoplakin, for instance, the surface cells covering the early mouse embryo not only lose desmosomal connections to keratin IFs, but also possess few desmosomes (3Gallicano I.G Kouklis P Bauer C Yin M Vasioukhin V Degenstein L Fuchs E J. Cell Biol. 1998; 143: 2009-2022Crossref PubMed Scopus (250) Google Scholar). Consequently, egg cylinder formation cannot progress, and the amniotic cavity fails to form. The failure of desmoplakin null embryos to survive most likely relates to their inability to withstand the stresses necessary to sculpt embryo shape. Similarly in somatic tissues, desmosomes are most abundant in heart muscle and epidermis, where mechanical wear and tear is of key concern. Finally, defects in cell type–specific desmosomal proteins result in degenerative disorders in humans, which in many respects resemble those involving IF mutations. The IF cytoskeleton has been employed not only for reinforcing and stabilizing cellular junctions, but also for integrating and bracing other cytoskeletal networks. Some plakins possess an actin-binding domain (ABD) enabling them to interconnect IF and actin cytoskeletons (1Andra K Nikolic B Stocher M Drenckhahn D Wiche G Genes Dev. 1998; 12: 3442-3451Crossref PubMed Scopus (151) Google Scholar, 19Yang Y Bauer C Strasser G Wollman R Julien J.P Fuchs E Cell. 1999; 98: 229-238Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). This is a feature of two sensory neuron splice forms of the BPAG1 gene, BPAG1n1 and BPAG1n2, which contain novel amino-terminal sequences that include an ABD. When cointroduced with neuronal IFs (NFs) into cultured cells lacking their own cytoplasmic IFs, BPAG1n1 aligns the NFs along actin stress fibers (19Yang Y Bauer C Strasser G Wollman R Julien J.P Fuchs E Cell. 1999; 98: 229-238Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar and references therein). Thus, rather than link keratin IFs to hemidesmosomes as BPAG1e can do, BPAG1n1 and BPAG1n2 may instead anchor NFs to the axonal actin cytoskeleton. Consistent with this notion, dystonia musculorum (dt/dt) mutant mice, defective in the BPAG1 gene, display spastic ataxic movements, accompanied by NF disorganization and sensory neuron degeneration. Plectin also has an ABD and has recently been implicated in cellular processes involving actin microfilament (MF) dynamics. Ironically, cultured plectin null fibroblasts are more adherent, less motile, and fail to display short-term rearrangements of MFs in response to Rho/Rac/Cdc42 activation (1Andra K Nikolic B Stocher M Drenckhahn D Wiche G Genes Dev. 1998; 12: 3442-3451Crossref PubMed Scopus (151) Google Scholar). Thus, rather than merely stabilizing the actin cytoskeleton through scaffolding it to the IF network, plectin may also orchestrate the dynamics of focal contact assembly and actin stress fiber formation, perhaps by organizing the regulatory molecules involved (1Andra K Nikolic B Stocher M Drenckhahn D Wiche G Genes Dev. 1998; 12: 3442-3451Crossref PubMed Scopus (151) Google Scholar). This further broadens the scope of possible functions for these cytoskeletal connectors. While linkage to IFs has always been assumed to be a key feature of plakins, several observations suggest that these proteins may possess functions independent of IF association. The most compelling evidence stems from the recent discovery of plakins in lower eukaryotes that lack IF cytoplasmic networks (5Gregory S.L Brown N.H J. Cell Biol. 1998; 143: 1271-1282Crossref PubMed Scopus (128) Google Scholar, 13Prokop A Uhler J Roote J Bate M J. Cell Biol. 1998; 143: 1283-1294Crossref PubMed Scopus (106) Google Scholar, 14Strumpf D Volk T J. Cell Biol. 1998; 143: 1259-1270Crossref PubMed Scopus (103) Google Scholar). Interestingly, despite the lack of a need for, or possession of, a conventional IF-binding domain, Drosophila kakapo plakin functions similarly to some mammalian plakins. kakapo mutants are defective in the integrin junctions between muscle and either neurons or epidermal cells. In larvae, ectodermally expressed kakapo is also required for polarization of Vein, which is secreted by adjacent myotubes and transmits a signal converting ectodermal cells to tendons (14Strumpf D Volk T J. Cell Biol. 1998; 143: 1259-1270Crossref PubMed Scopus (103) Google Scholar). How does kakapo function, and how does this help us to understand the roles of vertebrate plakins? In flies, integrin junctions appear to be stabilized by MTs, and correspondingly, kakapo mutant embryos display abnormalities in microtubule organization at sites where epidermal cells and neurons are detached from their underlying support muscle (5Gregory S.L Brown N.H J. Cell Biol. 1998; 143: 1271-1282Crossref PubMed Scopus (128) Google Scholar, 13Prokop A Uhler J Roote J Bate M J. Cell Biol. 1998; 143: 1283-1294Crossref PubMed Scopus (106) Google Scholar). This led to speculation that kakapo may stabilize the fly's integrins through linkage to actin and microtubule networks (Figure 1B). Intriguingly, while MFs have long been known to be critical for vertebrate integrin function, MTs were recently found to be concentrated at the integrin-rich focal contacts of vertebrate cells (8Kaverina I Rottner K Small J.V J. Cell Biol. 1998; 142: 181-190Crossref PubMed Scopus (250) Google Scholar). In fact, changes in MT dynamics have recently been found to affect MF assembly at the leading edge of migrating cells, thus further implicating an MT–actin connection not only in cell–substratum contacts, but also in migration (18Waterman-Storer C Salmon T Curr. Opin. Cell Biol. 1999; 11: 61-67Crossref PubMed Scopus (214) Google Scholar). Moreover, like kakapo, some vertebrate plakins associate with MTs. Notably, plectin antibodies decorate the ultrastructural bridges connecting MTs and IFs in actin-extracted fibroblasts in vitro (Figure 2; see also 15Svitkina T Verkhovsky A Borisy G J. Cell Biol. 1996; 135: 991-1007Crossref PubMed Scopus (319) Google Scholar). Considering plectin's potential for interacting with both MTs and actin, coupled with its localization to hemidesmosomes in epidermal cells, the parallels between integrin–cytoskeletal junctions of lower and higher eukaryotes become striking. An ironic twist for the plakin family is that some members seem to function predominantly through their ability to bind to MTs, making them quintessential microtubule-associated proteins or MAPs. BPAG1n3, a new sensory neural isoform, is a novel plakin that directly binds MTs in vivo and in vitro (19Yang Y Bauer C Strasser G Wollman R Julien J.P Fuchs E Cell. 1999; 98: 229-238Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Lacking the ABD, BPAG1n3 localizes specifically to MTs, and also renders them stable to depolymerizing agents. Conversely, sensory neurites cultured from BPAG1 single or BPAG1/NF double null mutant mice display short MTs that are both cold- and drug-sensitive (19Yang Y Bauer C Strasser G Wollman R Julien J.P Fuchs E Cell. 1999; 98: 229-238Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Taken together, these findings define BPAG1 plakins as integrators of all three cytoskeletal networks, able to brace and stabilize MT networks. MT stabilization is important for axons that transport cargo for distances up to a meter. Whether other plakins perform a similar function in other cell types, such as motor neurons, seems likely but awaits future studies. In lower and higher eukaryotic cells, various movements require coordination between actin and MT cytoskeletons. Such events include positioning the nucleus (particularly important in asymmetrically dividing cells), orienting the mitotic spindle with respect to the axis of cell division, shuttling vesicles and organelles between MT and actin-based transport systems, and guiding axonal growth cone movement. Positioning the nucleus and orienting the mitotic spindle have been extensively explored in budding yeast, where the site of the next cell division is specified at the start of the cell cycle by the bud site. Prior to mitosis, the nucleus must first transit along cytoplasmic MT tracks to a position near the bud neck. This process is motored by the plus end–directed motor, Kip3p kinesin, required to orient the spindle along the mother bud axis (6Heil-Chapdelaine R.A Adames N.R Cooper J.A J. Cell Biol. 1999; 144: 809-811Crossref PubMed Scopus (45) Google Scholar). Experiments with actin depolymerizing drugs suggest that spindle orientation requires transient attachment of MTs to the cortical actin cytoskeleton at the bud tip. Molecular details of this actin–MT connection are beginning to unfold, and mutations in the Bni1, Bud6, and Kar9 genes all affect spindle orientation (Figure 1C; 10Lee L Klee S.K Evangelista M Boone C Pellman D J. Cell Biol. 1999; 144: 947-961Crossref PubMed Scopus (129) Google Scholar, 11Miller R.K Matheos D Rose M.D J. Cell Biol. 1999; 144: 963-975Crossref PubMed Scopus (137) Google Scholar). A role for Bni1p is tantalizing since its higher eukaryotic cousins, formins, are known regulators of the actin cytoskeleton (10Lee L Klee S.K Evangelista M Boone C Pellman D J. Cell Biol. 1999; 144: 947-961Crossref PubMed Scopus (129) Google Scholar). Additionally, all three proteins localize to the actin patch at the bud tip, although direct evidence is still lacking that any of them binds to MTs. Another possible candidate for making this connection is the conserved coronin protein, able to bind to both actin and MTs in vitro and implicated in actin dynamics as well as MT organization in budding yeast (4Goode B.L Wong J.J Butty A.C Peter M McCormack A.L Yates J.R Drubin D.G Barnes G J. Cell Biol. 1999; 144: 83-98Crossref PubMed Scopus (183) Google Scholar). The connection of spindle to cell cortex is also utilized in directing movement of the anaphase spindle into the neck, once it has been oriented. Dynein, a minus end-directed MT motor, and dynactin, its activator, seem to associate more broadly with the cortex than the actin cap proteins. Dynein either pulls on cytoplasmic MTs or influences MT assembly dynamics to achieve this movement (6Heil-Chapdelaine R.A Adames N.R Cooper J.A J. Cell Biol. 1999; 144: 809-811Crossref PubMed Scopus (45) Google Scholar). Many of the lessons being learned from spindle movement and polarization in the yeast bud are likely to be operative in higher eukaryotes, given the crucial requirement for directed MT orientation and transport in processes ranging from epithelial stratification and tissue morphogenesis, to oogenesis and neuronal cell fate determination, to the transport of MTs into axons. Indeed dynein, dynactin, and formins have already been implicated in these processes in higher animals. Another universal process that relies upon actin–MT interactions is vesicle and organelle trafficking. A particularly fine example exists in neurons, where long-distance transport of vesicles out of the cell body begins by kinesin-motored trafficking along the extended MT highways of axons. At the growth cone, where axonal MTs end in a delta of MFs, vesicles must be relinquished to myosins, the actin-based motors. Yeast two-hybrid studies suggest that the vesicle may be transferred in baton-like fashion from kinesin (KhcU) to myosin MyoVA (7Huang J.D Brady S.T Richards B.W Stenolen D Resau J.H Copeland N.G Jenkins N.A Nature. 1999; 397: 267-270Crossref PubMed Scopus (276) Google Scholar). Located at the crossroads of these two highways are several other proteins, including the MT-associated protein MAP1B, abundant in the distal axons of cultured neurites, as well as in early postnatal brain regions that retain axonal growth activity and synaptic plasticity. Recently an actin-binding domain was uncovered in the MAP1B light chain, suggesting that it may act to bridge the two cytoskeletal highways (16Togel M Wiche G Propst F J. Cell Biol. 1998; 143: 695-707Crossref PubMed Scopus (128) Google Scholar). Additionally, the association of MAP1B with MFs depends upon MAP1B phosphorylation, providing a possible mechanism for regulating MT–actin connections in response to external or intracellular signals. Cytoskeletal highways can also be used to transport cytoskeletal components, as illustrated by the discovery of the motile and dynamic properties of IF networks mediated through interactions with MTs and MFs (20Yoon M Moir R.D Prahlad V Goldman R.D J. Cell Biol. 1998; 143: 147-157Crossref PubMed Scopus (200) Google Scholar). While a clear picture is just beginning to emerge for how the roadways and motors of the actin- and MT-based cytoskeleton are linked, an equally intriguing problem is how the traffic of vesicles and organelles is orchestrated. Researchers have now identified MAPs that have the added capacity to bind to endosomes, kinetochores, and other particles in cells. The prototype is human CLIP-170, an MT linker protein that binds preferentially to the growing, i.e. plus, ends of MTs, enabling these proteins to treadmill in partnership with the most dynamic MTs in the cytoplasm (12Perez F Diamantopoulos G.S Stalder R Kreis T.E Cell. 1999; 96: 517-527Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). CLIP-170 also interacts with the dynactin complex, perhaps acting as a capturing device to establish initial contact between a particle and MT, and then transfer the cargo to dynein for transport toward the MT organizing center (17Vaughan K.T Tynan S.H Faulkner N.E Echeverri C.J Vallee R.B J. Cell Sci. 1999; 112: 1437-1447Crossref PubMed Google Scholar). Because CLIP-170 is often located near the actin cortex, it seems likely that the role of this protein, or of the company that it keeps, may rely at least in some circumstances upon actin. In this regard, a Drosophila homolog, D-CLIP-190, interacts directly with an unconventional myosin, raising the possibility that CLIPs may capture and transfer cargo to both MT- and actin-based motors (9Lantz V.A Miller K.G J. Cell Biol. 1998; 140: 897-910Crossref PubMed Scopus (114) Google Scholar). The multiplicity of interactions among motors, vesicle/organelle-binding proteins, and actin and MT cytoskeletons may be a reflection of the Herculean tasks required of proteins that must direct congested traffic across complicated and dynamic actin–MT crossroads of the cytoskeletal transit system. As we face the millenium, a new and more integrated view of the cytoskeleton has begun to emerge. While each cytoskeleton retains its identity, the three networks rely heavily upon one another, employing connections for cytoskeletal stability, intracellular transport and trafficking, cytokinesis, cell polarity, and tissue morphogenesis. Key genetic evidence underscores the importance of connector proteins to cytoarchitecture and structural integrity as well as many dynamic cellular processes including migration. The emergence of plakins as a vital group of linker proteins that bridge IFs, MTs, and/or MF networks has paved the entrance to a remarkable interstate of novel functional interactions among the structural elements within the cytoplasm. The discovery of actin–MT connections as well as plakins in lower eukaryotes that lack IFs, suggest that the foundations for these fascinating new cellular pathways have ancient origins.