Satya Khuon

Microfilament-dependent movement of the beta3 integrin subunit within focal contacts of endothelial cells.

To gain insight into the dynamic properties of focal contacts, we induced expression of green fluorescent protein‐tagged β3 integrin (GFP‐β3) and actinin‐1 (GFP‐actinin‐1) in endothelial cells. Both tagged proteins localize with αvβ3 integrin in focal contacts distributed towards the periphery of transfected cells. Labeled focal contacts migrate at about 0.1 μm/min in stationary live endothelial cells. We compared β3 integrin and actinin‐1 dynamics in focal contacts by using fluorescence recovery after photobleaching. Recovery of signal in bleached focal contacts that have incorporated actinin‐1 is rapid and occurs within less than 4 min. This recovery is energy‐dependent. In contrast, recovery of bleached focal contacts that contain GFP‐β3 integrin takes longer than 30 min. Yet, when a narrow stripe of fluorescence is bleached across a β3 integrinlabeled focal contact, recovery is complete within 16 min. The latter recovery is energy‐dependent and is blocked not only by actin‐filament disrupting drugs but also by a myosin light chain kinase inhibitor. Thus, integrins are not immobile when incorporated into focal contacts, as some have suggested. We propose that integrins are mobile within the confines of focal contacts and that this mobility is supported by an actin‐associated molecular motor.

Myosin light chain kinase mediates transcellular intravasation of breast cancer cells through the underlying endothelial cells: a three-dimensional FRET study.

The transient and localized signaling events between invasive breast cancer cells and the underlying endothelial cells have remained poorly characterized. We report a novel approach integrating vascular engineering with three-dimensional time-lapse fluorescence resonance energy transfer (FRET) imaging to dissect how endothelial myosin light chain kinase (MLCK) is modulated during tumor intravasation. We show that tumor transendothelial migration occurs via both paracellular (i.e. through cell-cell junctions) and transcellular (i.e. through individual endothelial cells) routes. Endothelial MLCK is activated at the invasion site, leading to regional diphosphorylation of myosin-II regulatory light chain (RLC) and myosin contraction. Blocking endothelial RLC diphosphorylation blunts tumor transcellular, but not paracellular, invasion. Our results implicate an important role for endothelial myosin-II function in tumor intravasation.

BH3 Peptides Induce Mitochondrial Fission and Cell Death Independent of BAX/BAK

BH3 only proteins trigger cell death by interacting with pro- and anti-apoptotic members of the BCL-2 family of proteins. Here we report that BH3 peptides corresponding to the death domain of BH3-only proteins, which bind all the pro-survival BCL-2 family proteins, induce cell death in the absence of BAX and BAK. The BH3 peptides did not cause the release of cytochrome c from isolated mitochondria or from mitochondria in cells. However, the BH3 peptides did cause a decrease in mitochondrial membrane potential but did not induce the opening of the mitochondrial permeability transition pore. Interestingly, the BH3 peptides induced mitochondria to undergo fission in the absence of BAX and BAK. The binding of BCL-XL with dynamin-related protein 1 (DRP1), a GTPase known to regulate mitochondrial fission, increased in the presence of BH3 peptides. These results suggest that pro-survival BCL-2 proteins regulate mitochondrial fission and cell death in the absence of BAX and BAK.

Analysis of Cellular Localization and Function of Carboxy-Terminal Mutants of Pendrin

Background: Iodide uptake at the basolateral membrane and iodide efflux at the apical membrane of thyrocytes, essential steps in the biosynthesis of thyroid hormone, are stimulated by thyroid stimulating hormone (TSH). Pendrin (SLC26A4) is inserted into the apical membrane of thyrocytes and thought to be involved in mediating iodide efflux. Methods: We determined the effects of carboxy-terminal mutations of pendrin on the cellular localization and the ability to transport iodide. Results: After exposure to forskolin, the membrane abundance of wild type pendrin and iodide efflux increase. Truncation mutants lead to complete intracellular retention. Elimination of the distal part of the sulfate transporter and antisigma factor antagonist (STAS) domain with retention of the putative protein kinase A (PKA) phosphorylation site (RKDT 714-717) results in residual membrane insertion and a partial loss of function. Deletion of the PKA site results in decreased basal function and membrane insertion and abolishes the response to forskolin. Conclusion: Pendrin membrane abundance and its ability to mediate iodide efflux increase after activation of the PKA pathway. Elimination of the PKA site abolishes the response to forskolin but partial basal function and membrane insertion are maintained.

Dynamic aspects of cytoskeletal and karyoskeletal intermediate filament systems during the cell cycle

IF are major cytoskeletal and karyoskeletal components of eukaryotic cells (Steinert and Roop 1988). In numerous instances, their constituent protein subunits have been shown to be substrates for a variety of kinases such as A-kinase, C-kinase, and Ca++/calmodulin kinase (Geisler and Weber 1988; Inagaki et. 1988; Ando et al. 1991), as well as p34cdc2 (Chou et al. 1990; Peter et al. 1990; Ward and Kirschner 1990; Dessev et al. 1991). To date, all of the phosphorylation sites that have been mapped are in the non-alpha-helical amino- or carboxy-terminal domains (Steinert 1988; Ando et al. 1989, 1991; Geisler et al. 1989; Chou et al. 1991), and these secondary modifications can lead to IF reorganization and/or disassembly in vivo and in vitro (see, e.g., Iganaki et al. 1988; Lamb et al. 1989; Chou et al. 1990; Peter et al. 1990; Heald and McKeon 1990; Dessev et al. 1991). In addition, it is possible that the exchange seen between subunits and polymerized IF in interphase following the microinjection of unpolymerized protein (Vikstrom et al. 1989; Miller et al. 1991) may also be regulated in some fashion by phosphorylation/dephosphorylation reactions. In cultured fibroblasts such as BHK-21, the interphase equilibrium state that favors IF polymerization is shifted dramatically to a disassembled state in mitosis, apparently due to enhanced phosphorylation at specific sites mediated through the activity of p34cdc2. However, in other cells in mitosis, such as HeLa, the mechanisms involved in the regulation of cytoskeletal IF remain unclear. Therefore, no one common mechanism appears to be responsible for IF regulation during cell division. On the basis of the majority of data available, it appears that the regulation of IF phosphorylation plays an important role in the regulation of the supramolecular organization of IF cytoskeletal and karyoskeletal networks, especially in the remodeling events that take place as cells enter and exit mitosis. Although the functional significance of IF phosphorylation during interphase is not as obvious as it is in some mitotic cells, we are tempted to speculate that there may be a connection with mechanisms involved in signal transduction, since IF proteins appear to be targets for kinases known to be activated by second messengers such as Ca++ and cAMP.

Human Schlafen 5 (SLFN5) Is a Regulator of Motility and Invasiveness of Renal Cell Carcinoma Cells

ABSTRACT We provide evidence that human SLFN5, an interferon (IFN)-inducible member of the Schlafen (SLFN) family of proteins, exhibits key roles in controlling motility and invasiveness of renal cell carcinoma (RCC) cells. Our studies define the mechanism by which this occurs, demonstrating that SLFN5 negatively controls expression of the matrix metalloproteinase 1 gene (MMP-1), MMP-13, and several other genes involved in the control of malignant cell motility. Importantly, our data establish that SLFN5 expression correlates with a better overall survival in a large cohort of patients with RCC. The inverse relationship between SLFN5 expression and RCC aggressiveness raises the possibility of developing unique therapeutic approaches in the treatment of RCC, by modulating SLFN5 expression.

Structure and biomechanics of the endothelial transcellular circumferential invasion array in tumor invasion.

Cancer cells breach the endothelium not only through cell-cell junctions but also via individual endothelial cells (ECs), or transcellular invasion. The underlying EC forms a circular structure around the transcellular invasion pore that is dependent on myosin light chain kinase (MLCK) and myosin II regulatory light chain (RLC) phosphorylation. Here we offer mechanistic insights into transcellular invasive array formation amid persistent tensile force from activated EC myosin. Fluorescence recovery after photobleaching (FRAP) experiments, sarcomeric distance measurements using super-resolution microscopy and electron microscopy provide details about the nature of the myosin II invasion array. To probe the relationship between biomechanical forces and the tension required to maintain the curvature of contractile filaments, we targeted individual actin-myosin fibers at the invasion site for photoablation. We showed that adjacent filaments rapidly replace the ablat11ed structures. We propose that the transcellular circumferential invasion array (TCIA) provides the necessary constraint within the EC to blunt the radial compression from the invading cancer cell.

Intermediate Filament Cytoskeletal System: Dynamic and Mechanical Properties

Intermediate filaments (IF) are major cytoskeletal constituents of animal cells. For many years they were thought to be the most stable of all of the different cytoskeletal systems. Recently, however, IF have been shown to be in a state of dynamic equilibrium in growing cells. In fibroblasts and epithelial cells, this has been demonstrated in vivo by the finding that microinjected, soluble vimentin and keratin subunits are incorporated into endogenous IF (Vikstrom et al., 1989; Miller et al., 1991). Other experiments involving fluorescence recovery after photobleaching (FRAP) have demonstrated the existence of a steady-state equilibrium between polymerized IF and their subunits in vivo (Vikstrom et al., 1992). Furthermore, the exchange between IF subunits and their polymers appears to be regulated by phosphorylation catalyzed by different kinases and dephosphorylation catalyzed by phosphatases (Eriksson et al., 1992). Recently, we have exploited this equilibrium state in vivo through the use of mimetic peptides that are known to both drive vimentin IF disassembly and to inhibit subunit polymerization into IF at 1:1 molar ratios in vitro. The sequence of these peptides is derived from the helix 1A initiation domain of the central rod region of either keratin or vimentin. The same 1A peptide carrying a single point mutation has no obvious effect on vimentin IF assembly in vitro up to a 10-fold molar excess. We have also shown that there are no detectable effects of the wild-type pep-