Aberrant signaling through the class I phosphatidylinositol 3-kinase (PI3K)-Akt axis is frequent in human cancer. Here, we show that Beclin 1, an essential autophagy and tumor suppressor protein, is a target of the protein kinase Akt. Expression of a Beclin 1 mutant resistant to Akt-mediated phosphorylation increased autophagy, reduced anchorage-independent growth, and inhibited Akt-driven tumorigenesis. Akt-mediated phosphorylation of Beclin 1 enhanced its interactions with 14-3-3 and vimentin intermediate filament proteins, and vimentin depletion increased autophagy and inhibited Akt-driven transformation. Thus, Akt-mediated phosphorylation of Beclin 1 functions in autophagy inhibition, oncogenesis, and the formation of an autophagy-inhibitory Beclin 1/14-3-3/vimentin intermediate filament complex. These findings have broad implications for understanding the role of Akt signaling and intermediate filament proteins in autophagy and cancer.

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. Phosphorylation of lamin proteins is believed to cause lamina disassembly during meiotic and mitotic M phase, but the M phase-specific lamin kinase has not been identified. Here we show that the cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type lamins in vitro on sites that are specifically phosphorylated during M phase in vivo. Concomitantly, cdc2 kinase is capable of inducing lamina depolymerization upon incubation with isolated nuclei. One of the target sites of cdc2 kinase is identified as a motif (SPTR) conserved in the N-terminal domain of all lamin proteins. These results lead us to propose that mitotic disassembly of the nuclear lamina results from direct phosphorylation of lamins by cdc2 kinase.

The amino acid sequences deduced from cDNA clones of human lamin A and lamin C show identity between these two lamins except for an extra 9.0-kDa carboxyl-terminal tail that is present only in lamin A. Both lamins A and C contain an alpha-helical domain of approximately 360 residues that shows striking homology to a corresponding alpha-helical rod domain that is the structural hallmark of all intermediate filament proteins. However, the lamin alpha-helical domain is 14% larger than that of the intermediate filament proteins. In addition to the extensive homology to intermediate filament proteins as reported [McKeon, F., Kirschner, M. & Caput, D. (1986) Nature (London) 319, 463-468], a different 82-amino acid residue stretch at the carboxyl terminus of lamin A has been deduced and verified by amino acid sequencing. This region contains sequence homology to amino- and carboxylterminal domains of type I and type II epidermal keratins. Implications of the presence of these and other domains in lamins A and C for the assembly of the nuclear lamina are discussed.

Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.

Mutations in genes encoding the intermediate filament nuclear lamins and associated proteins cause a wide spectrum of diseases sometimes called "laminopathies." Diseases caused by mutations in LMNA encoding A-type lamins include autosomal dominant Emery-Dreifuss muscular dystrophy and related myopathies, Dunnigan-type familial partial lipodystrophy, Charcot-Marie-Tooth disease type 2B1 and developmental and accelerated aging disorders. Duplication in LMNB1 encoding lamin B1 causes autosomal dominant leukodystrophy and mutations in LMNB2 encoding lamin B2 are associated with acquired partial lipodystrophy. Disorders caused by mutations in genes encoding lamin-associated integral inner nuclear membrane proteins include X-linked Emery-Dreifuss muscular dystrophy, sclerosing bone dysplasias, HEM/Greenberg skeletal dysplasia and Pelger-Huet anomaly. While mutations and clinical phenotypes of "laminopathies" have been carefully described, data explaining pathogenic mechanisms are only emerging. Future investigations will likely identify new "laminopathies" and a combination of basic and clinical research will lead to a better understanding of pathophysiology and the development of therapies.

In most cell types intermediate or 10-mm filaments (IF) are a major cytoskeletal organization and, thus, directly or indirectly influence the structural appearance of the cytoplasm. In line with the cell type-specific expression patterns of different IF proteins in normal animal and human tissue, IF typing distinguishes the major tumor groups, as documented by results with several hundred human tumors classified by conventional histologic methods. Carcinomas are characterized by cytokeratins, sarcomas of muscle cells by desmin, nonmuscle sarcomas by vimentin, and gliomas by glial fibrillary acidic protein. Furthermore, certain tumors originating from the sympathetic nervous system, e.g., ganglioneuroblastoma, pheochromocytoma, and at least some neuroblastomas, are characterized by the presence of neurofilaments. Carcinomas can often be further subdivided with regard to their possible derivation by examining their cytokeratin profiles. The IF type characteristic of the cell of origin seems to be kept not only in the primary tumor but usually also in solid metastases. In general, tumors do not acquire additional IF types. Therefore, IF typing can provide an unambiguous and rapid characterization in certain cases, that are difficult to diagnose by conventional techniques. Some useful examples are the small cell tumors of childhood and the discrimination between undifferentiated carcinoma and lymphoma. IF typing of a few tumors has already led to a revision or reconsideration of the original light microscopic diagnosis. The combined results indicate that at least certain carcinomas, as well as certain other tumor types, seem to arise by the selective multiplication of a particular and identifiable cell type present in the normal tissue. The procedure is not restricted to tumor material. IF typing of Mallory bodies, Alzheimer's disease tangles, certain myopathies, and the cells of the amniotic fluid offers further interesting applications. Thus, IF typing should become a valuable new tool both in histology and surgical pathology.

The nuclear envelope links the cytoskeleton to structural components of the nucleus. It functions to coordinate nuclear migration and anchorage, organize chromatin, and aid meiotic chromosome pairing. Forces generated by the cytoskeleton are transferred across the nuclear envelope to the nuclear lamina through a nuclear-envelope bridge consisting of SUN (Sad1 and UNC-84) and KASH (Klarsicht, ANC-1 and Syne/Nesprin homology) proteins. Some KASH-SUN combinations connect microtubules, centrosomes, actin filaments, or intermediate filaments to the surface of the nucleus. Other combinations are used in cell cycle control, nuclear import, or apoptosis. Interactions between the cytoskeleton and the nucleus also affect global cytoskeleton organization. SUN and KASH proteins were identified through genetic screens for mispositioned nuclei in model organisms. Knockouts of SUN or KASH proteins disrupt neurological and muscular development in mice. Defects in SUN and KASH proteins have been linked to human diseases including muscular dystrophy, ataxia, progeria, lissencephaly, and cancer.

Vimentin is the major intermediate filament (IF) protein of mesenchymal cells. It shows dynamically altered expression patterns during different developmental stages and high sequence homology throughout all vertebrates, suggesting that the protein is physiologically important. Still, until recently, the real tasks of vimentin have been elusive, primarily because the vimentin-deficient mice were originally characterized as having a very mild phenotype. Recent studies have revealed several key functions for vimentin that were not obvious at first sight. Vimentin emerges as an organizer of a number of critical proteins involved in attachment, migration, and cell signaling. The highly dynamic and complex phosphorylation of vimentin seems to be a likely regulator mechanism for these functions. The implicated novel vimentin functions have broad ramifications into many different aspects of cell physiology, cellular interactions, and organ homeostasis.

Epithelial-to-mesenchymal transition (EMT) is a critical event in the progression toward cancer metastasis. The intermediate filament protein vimentin is an important marker of EMT and a requisite regulator of mesenchymal cell migration. However, it is not known how vimentin functionally contributes to cancer cell invasion. Here, we report that ectopic expression of oncogenic H-Ras-V12G and Slug induces vimentin expression and migration in pre-malignant breast epithelial cells. Conversely, vimentin expression is necessary for Slug- or H-Ras-V12G-induced EMT-associated migration. Furthermore, silencing of vimentin in breast epithelial cells results in specific changes in invasiveness-related gene expression including upregulation of RAB25 (small GTPase Rab25) and downregulation of AXL (receptor tyrosine kinase Axl), PLAU (plasminogen activator, urokinase) and ITGB4 (integrin β4-subunit). Importantly, gene expression profiling analyses reveal that vimentin expression correlates positively/negatively with these genes also in multiple breast cancer cell lines and breast cancer patient samples. Focusing on the tyrosine kinase Axl, we show that induction of vimentin by EMT is associated with upregulation of Axl expression and that Axl enhances the migratory activity of pre-malignant breast epithelial cells. Using null and knock-down cells and overexpression models, we also show that regulation of breast cancer cell migration in two- and three-dimensional matrices by vimentin is Axl- dependent and that Axl functionally contributes to lung extravasation of breast cancer cells in mice. In conclusion, our data show that vimentin functionally contributes to EMT and is required for induction of Axl expression. Moreover, these results provide a molecular explanation for vimentin-dependent cancer cell migration during EMT by identifying Axl as a key proximal component in this process.

The superfamily of intermediate filament (IF) proteins contains at least 65 distinct proteins in man, which all assemble into approximately 10 nm wide filaments and are principal structural elements both in the nucleus and the cytoplasm with essential scaffolding functions in metazoan cells. At present, we have only circumstantial evidence of how the highly divergent primary sequences of IF proteins lead to the formation of seemingly similar polymers and how this correlates with their function in individual cells and tissues. Point mutations in IF proteins, particularly in lamins, have been demonstrated to lead to severe, inheritable multi-systemic diseases, thus underlining their importance at several functional levels. Recent structural work has now begun to shed some light onto the complex fine tuning of structure and function in these fibrous, coiled coil forming multidomain proteins and their contribution to cellular physiology and gene regulation.

Vinculin, a protein associated with the cytoplasmic face of the focal adhesion plaques which anchor actin-containing microfilaments to the plasma membrane and attach a cell to the substratum, contains 8-fold more phosphotyrosine in cells transformed by Rous sarcoma virus than in uninfected cells. Because the transforming protein of RSV, p60src, is a protein kinase that modifies cellular proteins through the phosphorylation of tyrosine and because phosphotyrosine is a very rare modified amino acid, this result is a very rare modified amino acid, this result suggests that vinculin is a primary substrate of p60src. Only trace amounts of phosphotyrosine were detected in myosin heavy chains, alpha-actinin, filamin, and the intermediate filament protein vimentin. The modification of vinculin by p60src may be responsible in part for the disruption of the microfilament organization and for the changes in cell shape and adhesiveness which accompany transformation by Rous sarcoma virus.

An improved serum-free culture system has been developed for normal human epidermal keratinocytes (HK). Short-term clonal growth and differentiation studies are routinely performed in a defined medium consisting of optimized nutrient medium MCDB 153 supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, and phosphoethanolamine. A small amount of whole bovine pituitary extract (wBPE) is added for initiation of primary cultures, for frozen storage, and for serial culture. The need for feeder layers, conditioned medium, serum, and specialized culture surfaces has been eliminated entirely. With an optimal level of calcium ion (0.3 mM), colony-forming efficiency is about 30 percent and cellular multiplication rate is 0.96 doublings per day in the defined medium. A high-calcium concentration (1.0 mM) induces stratification and terminal differentiation, which can be quantified by counting cornified envelopes that are resistant to boiling in sodium dodecyl sulfate plus dithiothreitol. Under optimal conditions with wBPE present, cellular senescence occurs after about 40 population doublings. Scanning electron microscopy (SEM) has verified the occurrence of stratification during differentiation in the defined medium with high calcium. High-voltage electron microscopy (HVEM) after detergent extraction of human epidermal keratinocyte (HK) colonies grown in the defined medium with low and high calcium has revealed specific changes in the intermediate filament network and keratohyalin granules corresponding to changes in cellular differentiation. Indirect immunofluorescence studies have verified that the intermediate filament network observed with HVEM is composed of keratin proteins.

Keratins are intermediate filament-forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species.

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.

Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)-1 and -2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin alpha6beta4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.

Invasive cancer cells are believed to breach the basement membrane (BM) using specialized protrusions called invadopodia. We found that the crossing of a native BM is a three-stage process: invadopodia indeed form and perforate the BM, elongate into mature invadopodia, and then guide the cell toward the stromal compartment. We studied the remodeling of cytoskeleton networks during invadopodia formation and elongation using ultrastructural analysis, spatial distribution of molecular markers, and RNA interference silencing of protein expression. We show that formation of invadopodia requires only the actin cytoskeleton and filopodia- and lamellipodia-associated proteins. In contrast, elongation of invadopodia is mostly dependent on filopodial actin machinery. Moreover, intact microtubules and vimentin intermediate filament networks are required for further growth. We propose that invadopodia form by assembly of dendritic/diagonal and bundled actin networks and then mature by elongation of actin bundles, followed by the entry of microtubules and vimentin filaments. These findings provide a link between the epithelial to mesenchymal transition and BM transmigration.

We review the morphofunctional characteristics of pericytes and report our observations. After a brief historical background, we consider the following aspects of pericytes: A) Origin in embryonic vasculogenesis (mesenchymal stem cells, neurocrest and other possible sources) and in embryonic and postnatal life angiogenesis (pre-existing pericytes, fibroblast/ myofibroblasts and circulating progenitor cells). B) Location in pericytic microvasculature and in the other blood vessels (including transitional cell forms and absence in lymphatic vessels), incidence (differences depending on species, topographical location, and type and stage of vessels) and distribution (specific polarities) in blood vessels. C) Morphology (cell body, and longitudinal and circumferential cytoplasmic processes), structure (nucleus, cytoplasmic organelles and distribution of microtubules, intermediate filaments and microfilaments) and surface (caveolae system). D) Basement membrane disposition, formation, components and functions. E) Contacts with endothelial cells (ECs) (peg and socket arrangements, adherent junctions and gap junctions) and with basal membrane (adhesion plaques). F) Molecular expression (pericyte marker identification). G) Functions, such as vessel stabilization, regulation of vascular tone and maintenance of local and tissue homeostasis (contractile capacity and vessel permeability regulation), matrix protein synthesis, macrophage-like properties, immunological defense, intervention in coagulation, participation in mechanisms that regulate the quiescent and angiogenic stages of blood vessels (including the behaviour of pericytes during sprouting angiogenesis and intussuceptive vascular growth, as well as pericyte interactions with endothelium and other cells, and with extracellular matrix) and plasticity, as progenitor cells with great mesenchymal potential, originating other pericytes, fibroblast/myofibroblasts, preadipocytes, chondroblasts, osteoblasts, odontoblasts, vascular smooth muscle and myointimal cells. This mesenchymal capacity is seen in a broad section on the perivascular mesenchymal cell niche hypothesis and in the concept of pericyte and EC "marriage and divorce". H) Peculiar pericyte types, such as hepatic stellate cells (Ito cells), bone marrow reticular cells and mesangial cells. I) Involvement in pathological processes, such as repair through granulation tissue, pericyte-derived tumors, tumor angiogenesis and tumoral cell metastasis, diabetic microangiopathy, fibrosis, atherosclerosis and calcific vasculopathy, lymphedema distichiasis, chronic venous insufficiency, pulmonary hypertension, Alzheimer disease and multiple sclerosis. J) Clinical and therapeutic implications (de-stabilization of vessels or formation of a stable vasculature).

Intermediate filaments (IFs) constitute a major structural element of animal cells. They build two distinct systems, one in the nucleus and one in the cytoplasm. In both cases, their major function is assumed to be that of a mechanical stress absorber and an integrating device for the entire cytoskeleton. In line with this, recent disease mutations in human IF proteins indicate that the nanomechanical properties of cell-type-specific IFs are central to the pathogenesis of diseases as diverse as muscular dystrophy and premature ageing. However, the analysis of these various diseases suggests that IFs also have an important role in cell-type-specific physiological functions.

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP(-/-)Vim(-/-) mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP(-/-)Vim(-/-) than in wild-type (WT) mice; GFAP(-/-), Vim(-/-) and WT mice had the same infarct volume. Endothelin B receptor (ET(B)R) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP(-/-)Vim(-/-) astrocytes. In WT astrocytes, ET(B)R colocalized extensively with bundles of IFs. GFAP(-/-)Vim(-/-) astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP(-/-)Vim(-/-) than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET(B)R-mediated control of gap junctions, and PAI-1 expression.

In mouse embryos, acquisition of the nuclear lamin polypeptides A/C varies according to developmental stage and tissue type. In order to determine the precise time points and cell types in which lamin A/C are first observed, we have used two monoclonal antibodies in immunofluorescence studies of different tissues of developing mouse embryos and of young mice. One antibody (mAB346) is specific for lamins A and C, while the other (PKB8) detects lamins A, B and C. Dividing uterine development into three phases--germ layer formation, organogenesis and tissue differentiation--our results show that lamin A/C expression in the embryo proper is not observed until the third phase of development. Lamin A/C first appears at embryonic day 12 in muscle cells of the trunk, head and the appendages. Three days later it is also seen in cells of the epidermis where its appearance coincides with the time of stratification. In the simple epithelial of lung, liver, kidney and intestine, as well as in heart and brain, lamins A/C do not appear until well after birth. Embryonal carcinoma (EC) cells express lamin B but not lamin A/C. Lamin A/C expression is noted in some EC cells after they are induced to differentiate and in several differentiated teratocarcinoma cell lines. Our results suggest that commitment of a cell to a particular pathway of differentiation (assayed by cell-type-specific expression of intermediate filament proteins) usually occurs prior to the time that lamin A/C can be detected. Thus lamin A/C expression may serve as a limit on the plasticity of cells for further developmental events.

Spinal muscular atrophy (SMA) is a common pediatric neuromuscular disorder caused by insufficient levels of the survival of motor neuron (SMN) protein. Studies involving SMA patients and animal models expressing the human SMN2 gene have yielded relatively little information about the earliest cellular consequences of reduced SMN protein. In this study, we have used severe- and mild-SMN2 expressing mouse models of SMA as well as material from human patients to understand the initial stages of neurodegeneration in the human disease. We show that the earliest structural defects appear distally and involve the neuromuscular synapse. Insufficient SMN protein arrests the post-natal development of the neuromuscular junction (NMJ), impairing the maturation of acetylcholine receptor (AChR) clusters into 'pretzels'. Pre-synaptic defects include poor terminal arborization and intermediate filament aggregates which may serve as a useful biomarker of the disease. These defects are reflected in functional deficits at the NMJ characterized by intermittent neurotransmission failures. We suggest that SMA might best be described as a NMJ synaptopathy and that one promising means of treating it could involve maintaining function at the NMJ.

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament "branches" are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the "microtrabecular network" (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).