Fluorescent measurement of desmin intermediate filament assembly

Intermediate filaments (IF) are cytoskeletal elements that are believed to play a major role in the specification and maintenance of cell form. Although previously thought to be stable and static because of their relative insolubility in physiological solvents, IF have recently been shown to have dynamic properties not unlike those of other cytoskeletal elements. The methodology for measuring this dynamic behavior, however, has been mostly borrowed from studies of other filament proteins and are poorly suited to IF because of their unusual physicochemical properties. In this report we introduce a fluorescence assay for quantifying in vitro IF assembly. Desmin subunits labeled with iodoacetamidofluorescein (IAF) to approximately 0.4 mol/mol retain the ability to polymerize into filaments indistinguishable from unlabeled IF in the electron microscope. By spectrophotometry, however, up to 90% of the starting fluorescence is quenched upon maximal IF assembly from IAF-desmin subunits. This quench is proportional to the total concentration of desmin subunits and is a sensitive measure of the assembly process. The critical concentration of assembly, measured at 170 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.0, is 0.2 microM. This indicates that a significant level of unpolymerized desmin exists in steady-state equilibrium with polymerized filaments under these conditions and suggests that IF subunit-filament equilibria may play a role in cytoskeletal dynamics.     

Dissection of keratin dynamics: different contributions of the actin and microtubule systems

It has only recently been recognized that intermediate filaments (IFs) and their assembly intermediates are highly motile cytoskeletal components with cell-type- and isotype-specific characteristics. To elucidate the cell-type-independent contribution of actin filaments and microtubules to these motile properties, fluorescent epithelial IF keratin polypeptides were introduced into non-epithelial, adrenal cortex-derived SW13 cells. Time-lapse fluorescence microscopy of stably transfected SW13 cell lines synthesizing fluorescent human keratin 8 and 18 chimeras HK8-CFP and HK18-YFP revealed extended filament networks that are entirely composed of transgene products and exhibit the same dynamic features as keratin systems in epithelial cells. Detailed analyses identified two distinct types of keratin motility: (I) Slow (approximately 0.23 microm/min), inward-directed, continuous transport of keratin filament precursor particles from the plasma membrane towards the cell interior, which is most pronounced in lamellipodia. (II) Fast (approximately 17 microm/min), bidirectional and intermittent transport of keratin particles in axonal-type cell processes. Disruption of actin filaments inhibited type I motility while type II motility remained. Conversely, microtubule disruption inhibited transport mode II while mode I continued. Combining the two treatments resulted in a complete block of keratin motility. We therefore conclude that keratin motility relies both on intact actin filaments and microtubules and is not dependent on epithelium-specific cellular factors.     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

The internal affairs of an integrin

Integrins are adhesion receptors that exchange signals between the extracellular and intracellular compartments. From their cell surface transmembrane location, they interact with extracellular matrix ligands or cellular counter-receptors, translating external cues into signals that affect cytoskeletal organization, cell shape and motility. Conversely, intracellular events may modify the affinities of integrins for external ligands. Inside the cell, integrins connect with cytoskeletal structures that, until recently, were thought to be exclusively actin microfilaments. We comment on the case of the epithelial integrin, alpha(6)beta(4), which may instead interact with intermediate filaments. This integrin was recently shown by several laboratories to be part of the hemidesmosome complex, an epithelial adhesive structure that is the plasma membrane anchoring site for keratin-containing intermediate filaments.     

A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton

The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.     

Structural analysis of vimentin and keratin intermediate filaments by cryo-electron tomography

Intermediate filaments are a large and structurally diverse group of cellular filaments that are classified into five different groups. They are referred to as intermediate filaments (IFs) because they are intermediate in diameter between the two other cytoskeletal filament systems that is filamentous actin and microtubules. The basic building block of IFs is a predominantly alpha-helical rod with variable length globular N- and C-terminal domains. On the ultra-structural level there are two major differences between IFs and microtubules or actin filaments: IFs are non-polar, and they do not exhibit large globular domains. IF molecules associate via a coiled-coil interaction into dimers and higher oligomers. Structural investigations into the molecular building plan of IFs have been performed with a variety of biophysical and imaging methods such as negative staining and metal-shadowing electron microscopy (EM), mass determination by scanning transmission EM, X-ray crystallography on fragments of the IF stalk and low-angle X-ray scattering. The actual packing of IF dimers into a long filament varies between the different families. Typically the dimers form so called protofibrils that further assemble into a filament. Here we introduce new cryo-imaging methods for structural investigations of IFs in vitro and in vivo, i.e., cryo-electron microscopy and cryo-electron tomography, as well as associated techniques such as the preparation and handling of vitrified sections of cellular specimens.     

Dynamic properties of intermediate filaments: disassembly and reassembly during mitosis in baby hamster kidney cells

A morphological analysis of the organizational changes in the type III intermediate filament (IF) system in dividing baby hamster kidney (BHK-21) cells was carried out by immunofluorescence and immunoelectron microscopy. The most dramatic change occurred during prometaphase, when the typical network of long 10-nm-diameter IF characteristic of interphase cells disassembled into aggregates containing short 4-6 nm filaments. During anaphase-telophase, arrays of short IF reappeared throughout the cytoplasm, and, in cytokinesis, the majority of IF were longer and concentrated in a juxtanuclear cap. These results demonstrate that the relatively stable IF cytoskeletal system of interphase cells is partitioned into daughter cells during mitosis by a process of disassembly and reassembly. This latter process occurs in a series of morphologically distinct steps at different stages of the mitotic process.     

The role of the vimentin intermediate filaments in rat 3Y1 cells elucidated by immunoelectron microscopy and computer-graphic reconstruction

The three-dimensional arrangement of vimentin intermediate filaments (IF) was studied in 3Y1, rat fibroblastic cell line, to elucidate its biological role in the cell. While actin filaments were observed exclusively in the superficial part of the cell, vimentin IF were found to be abundantly present in the inside of the cell where microtubules were occasionally discovered. By whole-mount immunoelectron microscopy and computer-graphic reconstruction of serial thin sections, it was observed in more detail that vimentin IF are located very close to the nucleus, endoplasmic reticulum, and mitochondria. Vimentin IF were observed to be attached to these organelles laterally or terminally. Thus, we can reasonably assume that vimentin IF are major cytoskeletal structures deep inside the cell and that they play an important role in supporting the location of the organelles. This is the first report which has visualized the three-dimensional relationship between vimentin IF and the organelles of the cell.     

The role of the vimentin intermediate filaments in rat 3Y1 cells elucidated by immunoelectron microscopy and computer-graphic reconstruction

The three-dimensional arrangement of vimentin intermediate filaments (IF) was studied in 3Y1, rat fibroblastic cell line, to elucidate its biological role in the cell. While actin filaments were observed exclusively in the superficial part of the cell, vimentin IF were found to be abundantly present in the inside of the cell where microtubules were occasionally discovered. By whole-mount immunoelectron microscopy and computer-graphic reconstruction of serial thin sections, it was observed in more detail that vimentin IF are located very close to the nucleus, endoplasmic reticulum, and mitochondria. Vimentin IF were observed to be attached to these organelles laterally or terminally. Thus, we can reasonably assume that vimentin IF are major cytoskeletal structures deep inside the cell and that they play an important role in supporting the location of the organelles. This is the first report which has visualized the three-dimensional relationship between vimentin IF and the organelles of the cell.     

Ndel1 controls the dynein-mediated transport of vimentin during neurite outgrowth

Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1-6). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein alphaCOP. Ndel1 promotes the interaction between Lis1, alphaCOP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/alphaCOP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration.     

Vimentin intermediate filament and plectin provide a scaffold for invadopodia,facilitating cancer cell invasion and extravasation for metastasis

To investigate the molecular mechanisms of cancer metastasis, we have isolated a high-metastatic bladder cancer cell subpopulation from a low-metastatic cell line by using an in vivo selection system. Cells in the subpopulation showed a high ability to form invadopodia, the filamentous actin (F-actin)-based membrane protrusions that play an essential role in cancer cell invasion. Analysis of the gene expression profile revealed that the expression of an intermediate filament (IF) protein, vimentin and a cytoskeletal linker protein, plectin was up-regulated in the high-metastatic subpopulation compared with the low metastatic cell line. Here we report a novel role of vimentin IF and plectin in metastasis. In invasive bladder cancer cells, the vimentin IF-plectin-invadopodia F-actin link was formed. Disruption of this link severely impaired invadopodia formation, reducing the capacities of extracellular matrix degradation, transendothelial migration and metastasis. In addition, the vimentin assembly into the filaments was required for invadopodia formation. Our results suggest that plectin anchoring invadopodia to vimentin IF scaffolds and stabilizes invadopodia, which is a critical molecular process for cancer cell invasion and extravasation for metastasis.     

Steady state dynamics of intermediate filament networks

We have conducted experiments to examine the dynamic exchange between subunit and polymer of vimentin intermediate filaments (IF) at steady state through the use of xrhodamine-labeled vimentin in fluorescence recovery after photobleaching (FRAP) analysis. The xrhodamine-vimentin incorporated into the endogenous vimentin IF network after microinjection into fibroblasts and could be visualized with a cooled charge-coupled device (CCD) camera and digital imaging fluorescence microscopy. Bar shaped regions were bleached in the fluorescent IF network using a beam from an argon ion laser and the cells were monitored at various times after bleaching to assess recovery of fluorescence in the bleached zones. We determined that bleached vimentin fibers can recover their fluorescence over relatively short time periods. Vimentin fibers in living cells also can exhibit significant movements, but the recovery of fluorescence was not dependent upon movement of fibers. Fluorescence recovery within individual fibers did not exhibit any marked polarity and was most consistent with a steady state exchange of vimentin subunits along the lengths of IF.     

Intermediate filaments: versatile building blocks of cell structure

Cytoskeletal intermediate filaments (IF) are organized into a dynamic nanofibrillar complex that extends throughout mammalian cells. This organization is ideally suited to their roles as response elements in the subcellular transduction of mechanical perturbations initiated at cell surfaces. IF also provide a scaffold for other types of signal transduction that together with molecular motors ferries signaling molecules from the cell periphery to the nucleus. Recent insights into their assembly highlight the importance of co-translation of their precursors, the hierarchical organization of their subunits in the formation of unit-length filaments (ULF) and the linkage of ULF into mature apolar IF. Analyses by atomic force microscopy reveal that mature IF are flexible and can be stretched to over 300% of their length without breaking, suggesting that intrafilament subunits can slide past one another when exposed to mechanical stress and strain. IF also play a role in the organization of organelles by modulating their motility and providing anchorage sites within the cytoplasm.     

The bacterial cytoskeleton: an intermediate filament-like function in cell shape

Various cell shapes are encountered in the prokaryotic world, but how they are achieved is poorly understood. Intermediate filaments (IFs) of the eukaryotic cytoskeleton play an important role in cell shape in higher organisms. No such filaments have been found in prokaryotes. Here, we describe a bacterial equivalent to IF proteins, named crescentin, whose cytoskeletal function is required for the vibrioid and helical shapes of Caulobacter crescentus. Without crescentin, the cells adopt a straight-rod morphology. Crescentin has characteristic features of IF proteins including the ability to assemble into filaments in vitro without energy or cofactor requirements. In vivo, crescentin forms a helical structure that colocalizes with the inner cell curvatures beneath the cytoplasmic membrane. We propose that IF-like filaments of crescentin assemble into a helical structure, which by applying its geometry to the cell, generates a vibrioid or helical cell shape depending on the length of the cell.     

Role of phosphorylation on the structural dynamics and function of types III and IV intermediate filaments

Phosphorylation of types III and IV intermediate filaments (IFs) is known to regulate their organization and function. Phosphorylation of the amino-terminal head domain sites on types III and IV IF proteins plays a key role in the assembly/disassembly of IF subunits into 10 nm filaments, and influences the phosphorylation of sites on the carboxyl-terminal tail domain. These phosphorylation events are largely under the control of second messenger-dependent protein kinases and provide the cells a mechanism to reorganize the IFs in response to the changes in second messenger levels. In mitotic cells, Cdk1, Rho kinase, PAK1 and Aurora-B kinase are believed to regulate vimentin and glial fibrillary acidic protein phosphorylation in a spatio-temporal manner. In neurons, the carboxyl-terminal tail domains of the NF-M and NF-H subunits of heteropolymeric neurofilaments (NFs) are highly phosphorylated by proline-directed protein kinases. The phosphorylation of carboxyl-terminal tail domains of NFs has been suspected to play roles in forming cross-bridges between NFs and microtubules, slowing axonal transport and promoting their integration into cytoskeleton lattice and, in doing so, to control axonal caliber and stabilize the axon. The role of IF phosphorylation in disease pathobiology is discussed.     

The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells

Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.     

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks.     

Chasing Coiled Coils: Intermediate Filaments in Plants

The cytoskeleton's structurally most resilient components, the intermediate filaments (IFs), have attracted the interest of cell biologists for more than two decades. IFs form extensive networks in many animal cells, and are thought to provide considerable tensile strength to the cells and tissues. In fact, the term “cytoskeleton” has originally been coined for the insoluble fibrous remains of detergent extracted animal cells. Nevertheless, cells can survive quite well without an IF network, and even without the subunit proteins that build the 10 nm wide polymeric filaments. Hence, the vital function of these cytoskeletal components is still hotly debated. Against this background, it may be premature to start suggesting functions for IFs in plants. Yet this is exactly what quite a number of researchers have begun to do. Because much recent evidence supports the idea of a plant IF cytoskeleton, it seems timely to examine this evidence and discuss its impact on our current understanding of IF function.     

Ectopic expression of desmin in the epidermis of transgenic mice permits development of a normal epidermis

Cell architecture is largely based on the interaction of cytoskeletal proteins, which include intermediate filaments (IF), microfilaments, microtubules, as well as their type-specific membrane-attachment structures and associated proteins. In order to further our understanding of IF proteins and to address the fundamental issue whether different IF perform unique functions in different tissues, we expressed a desmin transgene in the basal epidermis of mice. Ectopic expression of desmin led to the formation of an additional, keratin-independent IF cytoskeleton and did not interfere with the keratin-desmosome interaction. We show that ectopic expression of a type III IF protein in basal keratinocytes did not interfere with the normal epidermal architecture and the program of terminal differentiation. This demonstrated that keratinocytes suffered no obvious detrimental effects from extra desmin filaments in their cytoplasm. In addition, we asked whether stable expression of desmin could rescue K5 null mice, which served as a model for severe EBS. Transgenic mice ectopically expressing desmin in the basal layer were mated with K5 heterozygous deficient animals to generate desmin rescue mice and analysed. In summary, our study support the notion that the different IF like desmin or keratins composing a IF network in vivo are central to cytoskeletal architecture and design in cells.