93 Intermediate ®laments and their associated proteins: multiple dynamic personalities Megan K Houseweart∗ and Don W Cleveland² A fusion of mouse and human genetics has now proven that regulated by phosphorylation, the recent identi®cation of intermediate ®laments form a ¯exible scaffold essential for several kinases involved strengthens the argument that structuring cytoplasm in a variety of cell contexts. In some these dynamics are true in vivo events. The growing cases, the formation of this scaffold is achieved through a list of intermediate-®lament-associated proteins (IFAPs) newly identi®ed family of intermediate-®lament-associated surely expands the repertoire of possible interactions proteins that form cross-bridges between intermediate permitted to IFs and may also add another layer of ®laments and other cytoskeletal elements, including actin and regulatory complexity. Indeed, the discovery of several microtubules. human and mouse diseases caused by mutations in the IFAP proteins plectin and neuronal bullous pemphigoid antigen (BPAG1n) illustrates the importance of these Addresses molecules in providing links between components of the ∗Ludwig Institute for Cancer Research and Division of Cellular and Molecular Medicine, University of California at San Diego, 9500 cellular cytoskeleton. Most would agree that intermediate Gilman Drive, La Jolla, CA 92093, USA ®laments can no longer be thought of as the least dynamic ²Ludwig Institute for Cancer Research, Division of Cellular and components of the cell cytoskeleton. This review focuses Molecular Medicine, and Department of Medicine and Neuroscience, on the recent advances in understanding IFs and their University of California at San Diego, 9500 Gilman Drive, La Jolla, CA associated proteins. 92093, USA; e-mail: [email protected] Current Opinion in Cell Biology 1998, 10:93±101 A growing family of vital intermediate http://biomednet.com/elecref/0955067401000093 ®lament cross-linkers Current Biology Ltd ISSN 0955-0674 Perhaps the most exciting new development in the IF ®eld has been the recognition that IFs have binding Abbreviations partners and that these partners that have important ALS amyotrophic lateral sclerosis BPAG bullous pemphigoid antigen functions in structuring the three-dimensional cytoplasm. GFAP glial ®brillary acidic protein The IF-associated proteins, or IFAPs as they have come IF intermediate ®lament to be called, are steadily gaining attention as more diseases IFAP IF-associated protein in humans and mice are shown to arise from mutations MD-EBS muscular dystrophy with epidermolysis bullosa NF neuro®lament in these proteins. The ability of IFAPs to link various NLS nuclear localization signal components of the cytoskeleton in many different cell SOD1 superoxide dismutase 1 types suggests several potential functions as dynamic regulators of cytoskeletal assembly and maintainers of IF network integrity. Support for this notion came recently Introduction from studies that utilized peptides corresponding to Intermediate ®laments (IFs) have long been thought a conserved helix region of IF proteins that, when of as ®xed structural bystanders around whom the injected into ®broblast cells, disrupted the IF, microtubule, •• lively activity of the cell is distributed. More recently, and micro®lament networks [1 ]. The authors proposed however, IFs and their associated proteins have been that the introduced peptides effectively competed for ®rmly established as constituents of deformable cellular IFAPs that would normally link the ®lament networks latticeworks, imparting integrity and strength to tissues together and in this way caused the rapid disassembly throughout the body. Long known to extend throughout of the cytoskeleton. Currently, the list of IFAPs includes the cytoplasm, possibly positioning the nucleus within the members that interact with IFs from all ®ve IF subtypes, cell, IF networks have been shown to reversibly link the but as new IFs (including the lens-speci®c beaded • plasma membrane to other cytoskeletal components to ®laments [2 ]) are identi®ed, the discovery of new linking modulate cell shape and confer resistance to mechanical partners will surely follow. stress. As dynamic components of the cytoplasmic and nuclear cytoskeletons, IFs are thought to contribute to Plectin: an essential linker between intermediate cellular structural rearrangements that occur during cell ®laments, microtubules, myosin, and actin division. Evidence for pools of soluble IF subunits that can One of the most thoroughly characterized IFAPs is plectin, exchange along the entire length of assembled ®laments an abundant and extremely large (>500 kDa) cytoskeletal has grown and now helps to explain the basis for the cross-linker. Plectin is expressed in many cell types and rapid changes in IF organization that observed in response was initially shown by solid phase binding analysis to to such stimuli as heat shock and application of growth interact with a wide variety of other proteins such as factors. As many examples of IF dynamics appear to be vimentin, glial ®brillary acidic protein, keratins, lamin B, 94 Cytoskeleton microtubule-associated proteins, α-spectrin, and all three attachment of epidermal cells to the basement membrane. neuro®lament proteins [3,4]. More recently, immunoelec- That the strength provided by plectin is an essential tron microscopy studies [5••] to evaluate plectin-binding feature of cellular architecture has been demonstrated by interactions revealed that a) plectin forms cross-bridges the recent discovery of mutations in the gene for plectin between IFs and microtubules, b) vimentin ®laments are as a cause of the human disease muscular dystrophy decorated with plectin projections, c) plectin links IFs with epidermolysis bullosa (MD-EBS) [13••±15••]. This and actin ®lament bundles, and d) plectin associates with inherited disease is characterized by muscle degeneration myosin ®laments in cultured cells. The extraordinarily and skin blistering due to a failure to anchor the cellular IF clear images of plectin cross-bridges between various network to the plasma membrane via hemidesmosomes. ®lament networks supports many of the previously Three of the four known mutations, an eight base postulated plectin interactions and is further corroborated pair insertion [13••], an eight base pair deletion [14••], by biochemical and domain sequence data. Inspection of and a single nucleotide deletion [15••], occur within the rat and human plectin sequences has demonstrated the same region of plectin and result in premature the existence of a putative amino-terminal actin-binding termination approximately one third of the way through domain [6]. the >500 kDa polypeptide. The fourth known mutation, a nine nucleotide deletion [15••], removes three amino acids Sequence inspection and mutagenesis studies have mapped from within a 23 amino acid stretch that was shown to be a nuclear localization signal (NLS) within plectin's carboxy- identical between human and rat plectin. terminal vimentin binding domain motif [7••]. Beyond its role as a versatile cross-linker in interphase, the Tissues from patients harboring the three truncation presence of a plectin NLS [6], the fact that plectin mutations are typically devoid of plectin, whereas the binds lamin B in vitro [4], and the established pattern fourth mutation apparently encodes an otherwise full of plectin disassociation from vimentin upon entry into length protein and results in reduced plectin levels. mitosis [8•] suggest several possible roles for plectin during Patients with any of the four mutations typically display cell division. One scenario predicts that separating from reduced levels of BPAG1e, muscle ®ber abnormalities, cytoplasmic IFs would free plectin and collapse the IF and hemidesmosomes without an inner attachment plate network into the typical cagelike IF structure seen during [13••±15••]. mitosis. Thus freed, the uncovered plectin NLS could sequester NLS binding proteins. Alternatively, plectin's In order to determine more directly which aspects association with lamin B may act to disassemble the of plectin cross-linking activities are essential in vivo, nuclear matrix during nuclear envelope breakdown, or transgenic mice were engineered that lack the plectin perhaps the interaction really functions in the opposite protein [16••]. These plectin-null mice die two to three manner, that is, to promote nuclear reassembly (as argued days after birth and display pathological features typical in detail [9]). Although these ideas are as yet only of MD-EBS, with a few notable differences. For example, speculative, the protein kinase p34cdc2 has been shown the keratinocytes of MD-EBS skin rupture at the basal to phosphorylate plectin and cause its dissociation from hemidesmosome level, whereas the keratinocytes of vimentin during mitosis [8•]. Similarly, phosphorylation plectin-null mice rupture at all levels and contain ul- of plectin by protein kinase A or C can inhibit the trastructurally normal hemidesmosomes and desmosomes. binding of plectin to lamin B [4], providing the means by Plectin-de®cient skeletal muscle cells in mice were often which plectin could accomplish the mitosis-speci®c roles necrotic and disrupted sarcomeres were prevalent, whereas proposed above. cardiomyocytes displayed abnormally arranged sarcomeres and disintegrating intercalated discs. Additionally, the Comparison of plectin's structural domains with those distribution and expression levels of selected cytoskeletal