R864 Dispatch

Cytoskeletal linkers: New MAPs for old destinations Megan K. Houseweart*† and Don W. Cleveland*†‡§

A new isoform of the – linker as ‘bullous pemphigoid antigen’ (BPAG). These BPAG has been found that binds to and stabilizes are large α-helical coiled-coil molecules which have axonal . This and other newly identified binding domains for one or more of the three cytoskele- -associated proteins are likely to be just the tal components (Figure 1). For example, the widely tip of an iceberg of multifunctional proteins that expressed, > 500 kD protein has been shown to stabilize and crosslink cytoskeletal filament networks. associate with microtubules, intermediate filaments (glial fibrillary acidic protein, , , all Addresses: *Ludwig Institute for Cancer Research, †Program in Biomedical Sciences, ‡Division of Cellular and Molecular Medicine and three neurofilament subunit proteins), actin, and §Department of Neuroscience, University of California at San Diego, itself [3]. Given the widespread distribution and multi- La Jolla, California 92093, USA. ple interactions that are characteristic of these proteins, E-mail: [email protected] it is not surprising that a number of human and mouse Current Biology 1999, 9:R864–R866 diseases have been attributed to aberrant or missing cross-linking proteins [4]. 0960-9822/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. This is the case for mice lacking the encoding the numerous isoforms of the essential ~280 kDa linker The of most eukaryotic cells contains a dynamic protein BPAG. Two neuronal isoforms of BPAG both have filamentous protein scaffold composed of 25 nm micro- a carboxy-terminal intermediate-filament-binding domain tubules, 4 nm actin filaments and 10 nm intermediate fila- and also an amino-terminal actin-binding region (Figure 1). ments. Together, this network of proteins is responsible for Both of these neuronal forms of BPAG can functionally the structural integrity of the , as well as for cell motility link actin filaments and neuronal intermediate filaments and adhesion and the intracellular transport of specific — — in vivo (although some evidence cargoes. Although these three components of the cytoskele- has questioned the universality of this interaction [5]). ton are often thought of and studied as distinct systems, a What is not in doubt is that without the neuronal forms growing body of evidence demonstrates that they are not of BPAG [6,7], mice develop a fatal sensory neuron disease only physically interconnected by cross-linking proteins, with a pathology consisting of abnormal axonal myelination but that the properties of these filamentous networks can and large accumulations of disordered neurofilaments and be modulated by linker proteins, perhaps explaining their organelles within axonal swellings. often coordinated actions in cells [1,2]. But there is more to this linker story. Subsequent This emerging family of sequence-related cross-linker pro- examination of the large sensory neurons in the BPAG- teins, named , includes plectin, ACF7, , deficient mice revealed a disorganized axonal micro- the and the many forms of a protein known tubule array [8]. In normal axons, neurofilaments,

Figure 1

Current members of the family of axonal 31 251 1561 2195 3290 cytoskeletal linker proteins. Protein binding ABD MT HeadRod IF binding BPAG1n1 domains and structural elements are shown: ABD, actin-binding domain; MT, microtubule- binding domain; IF binding, intermediate ABD MT HeadRod IF binding BPAG1n2 filament-binding domain. Cross hatched regions indicate possible masked or 32 145 965 13292237 2870 incomplete binding domains. The numbers ABD MT HeadRod IF binding BPAG1n3 indicate protein residue count. Structures of BPAG1n1 and BPAG1n2 are from [7]; that of 77 307 BPAG1n3 is from [1]; that of ACF71–3 is from [13]; and that of plectin is from [14]. ABDHeadRod IF binding ACF71-3

282 947 2070 4140 ABD Head Rod IF binding Plectin

Current Biology Dispatch R865

Figure 2

The crossbridging of microtubules, actin filaments and intermediate filaments by linker proteins such as BPAG and plectin creates a three-dimensional cytoplasmic scaffold that structures the neuronal axoplasm in a parallel array. Actin Microtubule

BPAG 1n3 Microtubule-binding domain

BPAG 1n1/BPAG 1n2 Actin-binding domain

Plectin Intermediate-filament-binding domain Current Biology

organelles and other cargoes from the nerve cell body are for the disorganization of microtubules observed in transported along microtubule tracks by motor proteins BPAG-deficient animals. to and from the nerve terminal. In the affected axons of the BPAG-deficient mice, it seemed likely that disrup- Instead, the answer apparently lies with yet another tion of the microtubule network had impeded axonal BPAG isoform, BPAG1n3, encoded by an alternatively transport, causing axonal accumulations of cargoes and spliced RNA transcript of the same BPAG . In this eventually neuronal death by strangulation. A plausible newly found isoform, the amino-terminal actin-binding hypothesis, perhaps, but why would the removal of a domain is replaced by a microtubule-binding domain cross-linker protein responsible for bridging actin fila- (Figure 1). Not only can this molecule crosslink — and ments and neuronal intermediate filaments result in a thereby align — intermediate filaments along micro- disrupted microtubule network? tubules, it is an authentic microtubule-associated protein (MAP) which strongly stabilizes microtubules against Fuchs and colleagues [1] have recently provided a drug-induced or low-temperature-induced disassembly (as convincing solution to this dilemma. To determine seen using in vitro microtubule binding assays and in whether the neurofilaments left untethered in the primary sensory neuronal cultures). absence of BPAG directly contribute to the disrupted- microtubule phenotype of BPAG-deficient axons, this All of this evidence combines to highlight two lessons. group examined the microtubule arrays of sensory axons First, proteins that link cytoskeletal elements are impor- in the absence of both BPAG and neurofilaments. tant components with essential structural roles in organiz- Although the microtubule networks of neurofilament- ing cytoplasm (highlighted for axoplasm in Figure 2). deficient mice were found to be aligned normally, the Each crossbridge is probably weak and reversible, but the mice lacking both neurofilaments and the BPAG linker combination of many weak links between filaments with proteins displayed disorganized microtubules and markedly different viscoelastic properties yields a compos- sensory neuron degeneration indistinguishable from the ite network with mechanical properties able to withstand BPAG-deficient mice. This finding strongly argues that large internal or external stresses. Second, crosslinking per neither untethered neurofilaments nor the absence of se may not be the most important functional property of at linkage between neurofilaments and actin is to blame least some of these linkers. In the case of BPAG1n3, what R866 Current Biology Vol 9 No 22

may come as a surprise to many is that, although it was dis- expected as well; for example, yeast genetics has recently covered in an unconventional manner, this MAP is one of identified coronin, for which homologues are known in the only known microtubule-binding proteins capable of Caenorhabditis elegans, mice and humans, as a potential conferring both drug and temperature stability on micro- cross-linker of actin and microtubule networks [12]. tubule networks in vivo. The two most widely studied Coronin was originally identified as a binding protein neuronal MAPs, tau and MAP2, cannot do this. capable of promoting the assembly and bundling of actin, but the most recent studies have demonstrated micro- Indeed, it should now be painfully obvious to the tubule defects in yeast with mutant, missing or over- microtubule mafia — including the senior author of this expressed coronin [12]. On the basis of what we know, it dispatch — that the method used for 25 years to identify seems safe to predict that these newly discovered proteins neuronal MAPs relied on contorted logic, to say the least. are revealing but the tip of the iceberg. In time, this Despite the long known stability of a large percentage of emerging family of cytoskeletal linkers and stabilizers will neuronal microtubules, all of the early MAPs were identi- certainly grow in number and importance. fied as non- components of microtubules purified after discarding the cold stable ones! Thus, the widely References expressed neuronal MAPs, such as tau and MAP2, were at 1. Yang Y, Bauer C, Strasser G, Wollmann R, Julien J-P, Fuchs E: Integrators of the that stabilize microtubules. Cell the outset unlikely to represent the repertoire of impor- 1999, 98:229-238. tant MAPs. Indeed, the early mortality of the BPAG- 2. Andra K, Nikolic B, Stocher M, Drenckhahn D, Wiche G: Not just scaffolding: plectin regulates actin dynamics in cultured cells. deficient mouse shows that removing factors that confer Dev 1998, 12:3442-3451. extreme microtubule stability can have more detrimental 3. Svitkina T, Verkhovsky A, Borisy G: Plectin sidearms mediate effects than eliminating proteins such as tau (the absence interaction of intermediate filaments with microtubules and other components of the cytoskeleton. J Cell Biol 1996, 135:991-1007. of which causes little or no discernible phenotype [9]). 4. Fuchs E, Cleveland DW: A structural scaffolding of intermediate filaments in health and disease. Science 1998, 279:514-519. Several factors converge on a common view that there 5. Leung CL, Sun D, Liem RKH: The intermediate filament protein is the specific interaction partner of mouse BPAG1-n must be many more, as yet undiscovered MAPs. First, () in neurons. J Cell Biol 1999, 144:435-446. there is the narrow range of expression of BPAG in 6. Guo L, Degenstein L, Dowling J, Yu Q-C, Wollmann R, Perman B, Fuchs E: Gene targeting of BPAG1: abnormalities in mechanical neurons. The clear implication of BPAG’s confinement strength and cell migration in stratified epithelia and neurologic to sensory neurons is that other classes of neurons escape degeneration. Cell 1995, 81:233-243. the need for BPAG by reliance on other, functionally 7. Yang Y, Dowling J, Yu Q-C, Kouklis P, Cleveland DW, Fuchs E: An essential cytoskeletal linker protein connecting actin similar microtubule crosslinkers and/or stabilizers. to intermediate filaments. Cell 1996, 86:655-665. Second, a quick search of the current sequence libraries 8. Denarier E, Fourest-Lieuvin A, Bosc C, Pirollet F, Chapel A, Margolis RL, Job D: Non-neuronal isoforms of STOP protein are reveals multiple proteins with long, coiled coils linked to responsible for microtubule cold stability in mammalian domains with sequence similarity to the intermediate- fibroblasts. Proc Natl Acad Sci USA 1998, 95:6055-6060. filament-binding region of BPAG. Third, and perhaps 9. Harada A, Oguchi K, Okabe S, Kuno J, Terada S, Ohshima T, Sato-Yoshitake R, Takei Y, Noda T, Hirokawa N: Altered microtubule most important, human genetics has recently identified organization in small calibre axons of mice lacking . Doublecortin as an essential 40 kDa MAP required for Nature 1994, 369:488-491. neuronal migration in development of the cortex [10,11]. 10. Gleeson JG, Lin PT, Flanagan LA, Walsh CA: Doublecortin is a microtubule-associated protein and is expressed widely by Doublecortin is restricted in expression to migrating migrating neurons. Neuron 1999, 23:257-271. neurons, and its absence results in the ‘double cortex 11. Francis F, Koulakoff A, Boucher D, Chafey P, Schaar B, Vinet M-C, Friocourt G, McDonnell N, Reiner O, Kahn A, et al.: Doublecortin is syndrome’ or ‘subcortical laminar heterotopia’, in which a developmentally regulated, microtubule associated protein neuronal migration in the cortex is severely disrupted. expressed in migrating and differentiating neurons. Neuron Like BPAG1n3, Doublecortin binds to and stabilizes 1999, 23:247-256. 12. Goode BL, Wong JJ, Butty A-C, Peter M, McCormack AL, Yates JR, microtubules against drug-induced or cold-induced dis- Drubin DG, Barnes G: Coronin promotes the rapid assembly and assembly. This finding highlights the point that, at a cross-linking of actin filaments and may link the actin and minimum, a careful examination of proteins associated microtubule in yeast. J Cell Biol 1999, 144:83-98. 13. Bernier G, Mathieu M, Repentigny Y, Vidal S, Kothary R: Cloning and with the pool of cold-stable microtubules is long overdue. characterization of mouse ACF7, a novel member of the dystonin subfamily of actin binding proteins. Genomics 1996, 38:19-29. 14. Green K, Virata M: Comparative structural analysis of The existence of cytoskeleton-associated proteins, , bullous pemphigoid antigen and plectin: members especially those that can crosslink filaments of different of a new gene family involved in organization of intermediate classes and affect filament dynamics and stability, is likely filaments. Int J Biol Macromol 1992, 14:145-153. to be a general property. Plectin, which can bridge all three filament classes, has been found to be necessary for short- term remodeling of actin arrays in response to extracellular stimuli, clearly implicating this enormous, multi-functional crosslinking molecule as a mediator of actin assembly dynamics [2]. And new actin-linking proteins are to be