Vimentin is used widely as a marker of the epithelial to mesenchymal transitions (EMTs) that take place during embryogenesis and metastasis, yet the functional implications of the expression of this type III intermediate filament (IF) protein are poorly understood. Using form factor analysis and quantitative Western blotting of normal, metastatic, and vimentin-null cell lines, we show that the level of expression of vimentin IFs (VIFs) correlates with mesenchymal cell shape and motile behavior. The reorganization of VIFs caused by expressing a dominant-negative mutant or by silencing vimentin with shRNA (neither of which alter microtubule or microfilament assembly) causes mesenchymal cells to adopt epithelial shapes. Following the microinjection of vimentin or transfection with vimentin cDNA, epithelial cells rapidly adopt mesenchymal shapes coincident with VIF assembly. These shape transitions are accompanied by a loss of desmosomal contacts, an increase in cell motility, and a significant increase in focal adhesion dynamics. Our results demonstrate that VIFs play a predominant role in the changes in shape, adhesion, and motility that occur during the EMT.

Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition

Actin cortex mechanics and cellular morphogenesis.

Novel functions of vimentin in cell adhesion, migration, and signaling.

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation--such light can be phototoxic to some biological systems. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 degrees C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.

Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

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.

INTERMEDIATE FILAMENTS: Molecular Structure, Assembly Mechanism, and Integration Into Functionally Distinct Intracellular Scaffolds

Intermediate filaments, actin-containing microfilaments and microtubules are the three main cytoskeletal systems of vertebrate and many invertebrate cells. Although these systems are composed of distinctly different proteins, they are in constant and intimate communication with one another. Understanding the molecular basis of this cytoskeletal crosstalk is essential for determining the mechanisms that underlie many cell-biological phenomena. Recent studies have revealed that intermediate filaments and their associated proteins are important components in mediating this crosstalk.

Intermediate filaments mediate cytoskeletal crosstalk

Recent evidence showing that intermediate filaments (IFs) are dynamic, motile elements of the cytoskeletal repertoire of vertebrate cells has overturned the long-standing view that they simply form static 'space filling' cytoplasmic networks In fact, many types of IF are now known to engage in a remarkable array of movements that are closely associated with their assembly, disassembly and subcellular organization Some of these motile properties are intrinsic to IFs and others are attributable to molecular crosstalk with either microtubules or actin-containing microfilaments This crosstalk is, to a large extent, mediated by molecular motors, including conventional kinesin and cytoplasmic dynein These motors are responsible for the high-speed delivery of nonfilamentous IF precursors and short filaments to specific regions of the cytoplasm, where they assemble into long IFs Interestingly, the patterns and speeds of IF movements vary in different cell types and even within different regions of the same cell These differences in motility may be related to their interactions with different types of molecular motor and/or other factors, such as IF-associated proteins

/pdf/intermediate-filaments-are-dynamic-and-motile-elements-of-1olurjzi4r.pdf

Intermediate filaments are dynamic and motile elements of cellular architecture.

▪ Abstract For many years, cytoplasmic intermediate filaments (IFs) were considered to be stable cytoskeletal elements contributing primarily to the maintenance of the structural and mechanical integrity of cells. However, recent studies of living cells have revealed that IFs and their precursors possess a remarkably wide array of dynamic and motile properties. These properties are in large part due to interactions with molecular motors such as conventional kinesin, cytoplasmic dynein, and myosin. The association between IFs and motors appears to account for much of the well-documented molecular cross talk between IFs and the other major cytoskeletal elements, microtubules, and actin-containing microfilaments. Furthermore, the associations with molecular motors are also responsible for the high-speed, targeted delivery of nonfilamentous IF protein cargo to specific regions of the cytoplasm where they polymerize into IFs. This review considers the functional implications of the motile properties of IFs and...

The dynamic and motile properties of intermediate filaments.

The role of the intranuclear movement of chromatin in gene expression is not well-understood. Herpes simplex virus forms replication compartments (RCs) in infected cell nuclei as sites of viral DNA replication and late gene transcription. These structures develop from small compartments that grow in size, move, and coalesce. Quantitative analysis of RC trajectories, derived from 4D images, shows that most RCs move by directed motion. Directed movement is impaired in the presence of actin and myosin inhibitors as well as a transcription inhibitor. In addition, RCs coalesce at and reorganize nuclear speckles. Lastly, distinct effects of actin and myosin inhibitors on viral gene expression suggest that RC movement is not required for transcription, but rather, movement results in the bridging of transcriptionally active RCs with nuclear speckles to form structures that enhance export of viral late mRNAs.

https://www.pnas.org/content/pnas/108/21/E136.full.pdf

Herpesviral replication compartments move and coalesce at nuclear speckles to enhance export of viral late mRNA

We have been able to observe the dynamic interactions between a specific messenger RNA (mRNA) and its protein product in vivo by studying the synthesis and assembly of peripherin intermediate filaments (IFs). The results show that peripherin mRNA-containing particles (messenger ribonucleoproteins [mRNPs]) move mainly along microtubules (MT). These mRNPs are translationally silent, initiating translation when they cease moving. Many peripherin mRNPs contain multiple mRNAs, possibly amplifying the total amount of protein synthesized within these “translation factories.” This mRNA clustering is dependent on MT, regulatory sequences within the RNA and the nascent protein. Peripherin is cotranslationally assembled into insoluble, nonfilamentous particles that are precursors to the long IF that form extensive cytoskeletal networks. The results show that the motility and targeting of peripherin mRNPs, their translational control, and the assembly of an IF cytoskeletal system are linked together in a process we have termed dynamic cotranslation.

/pdf/assembling-an-intermediate-filament-network-by-dynamic-1kw9pzl5rs.pdf

Lynne Chang

Papers

Intermediate filaments mediate cytoskeletal crosstalk

Intermediate filaments are dynamic and motile elements of cellular architecture.

The dynamic and motile properties of intermediate filaments.

Herpesviral replication compartments move and coalesce at nuclear speckles to enhance export of viral late mRNA

Assembling an intermediate filament network by dynamic cotranslation