Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells

Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non- cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum- deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker, consistent with the formation of a gradient of actin filament density relative to cell volume. Comparison of these gradients in the same living cell using analogs of actin with high and low affinities for profilin demonstrated that increased profilin binding enhanced the gradient. Profilin and related proteins may therefore function in part to bias the assembly of actin at the membrane-cytoplasm interface.     

A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells.

Phosphorylation of the regulatory light chain by myosin light chain kinase (MLCK) regulates the motor activity of smooth muscle and nonmuscle myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin II regulatory light chain phosphorylation (FRLC-Rmyosin II) is described here. The biosensor depends upon energy transfer from fluorescein-labeled regulatory light chains to rhodamine-labeled essential and/or heavy chains. The energy transfer ratio increases by up to 26% when the regulatory light chain is phosphorylated by MLCK. The majority of the change in energy transfer is from regulatory light chain phosphorylation by MLCK (versus phosphorylation by protein kinase C). Folding/unfolding, filament assembly, and actin binding do not have a large effect on the energy transfer ratio. FRLC-Rmyosin II has been microinjected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing FRLC-Rmyosin II with the kinase inhibitor staurosporine produced a lower ratio of rhodamine/fluorescein emission, which corresponds to a lower level of myosin II regulatory light chain phosphorylation. Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers.     

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.     

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may be performed. Non-muscle myosin IIA was purified from bovine platelets, non-muscle myosin IIB from bovine brain, and smooth muscle myosin II from turkey gizzards. After being fluorescently labeled with tetramethylrhodamine-5-iodoacetamide or with a succinimidyl ester of cy5, they retained the following properties: (1) reversible assembly into thick filaments, (2) actin-activatable MgATPase, (3) phosphorylation by myosin light chain kinase, (4) increased MgATPase upon light-chain phosphorylation, (5) interconversion between 6S and 10S conformations, and (6) distribution into endogenous myosin II-containing structures when microinjected into cultured cells. These fluorescent analogues can be used to visualize isoform-specific dynamics of myosin II in living cells. J. Cell. Biochem. 68:389–401, 1998. © 1998 Wiley-Liss, Inc.     

The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts

The formation of protrusions at the leading edge of the cell is an essential step in fibroblast locomotion. Using fluorescent analogue cytochemistry, ratio imaging, multiple parameter analysis, and fluorescence photobleaching recovery, the distribution of actin and myosin was examined in the same protrusions at the leading edge of live, locomoting cells during wound-healing in vitro. We have previously defined two temporal stages of the formation of protrusions: (a) initial protrusion and (b) established protrusion (Fisher et al., 1988). Actin was slightly concentrated in initial protrusions, while myosin was either totally absent or present at extremely low levels at the base of the initial protrusions. In contrast, established protrusions contained diffuse actin and actin microspikes, as well as myosin in both diffuse and structured forms. Actin and myosin were also localized along concave transverse fibers near the base of initial and established protrusions. The dynamics of myosin penetration into a relatively stable, established protrusion was demonstrated by recording sequential images over time. Myosin was shown to be absent from an initial protrusion, but diffuse and punctate myosin was detected in the same protrusion within 1-2 min. Fluorescence photobleaching recovery indicated that myosin was 100% immobile in the region behind the leading edge containing transverse fibers, in comparison to the 21% immobile fraction detected in the perinuclear region. Possible explanations of the delayed penetration of myosin into established protrusions and the implications on the mechanism of protrusion are discussed.     

Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils

To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.     

Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.     

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.     

Formation and movement of myosin-containing structures in living fibroblasts

Gizzard myosin, fluorescently labeled with tetramethylrhodamine iodoacetamide, was microinjected into living 3T3 fibroblasts to label myosin-containing structures. The fluorophore was located predominantly on the heavy chain near the COOH terminus of the S1 head and on the 17-kD light chain. After microinjection of a tracer amount into living 3T3 cells, the fluorescent myosin showed a distribution identical to that revealed by immunofluorescence with antimyosin antibodies. Injected myosin became localized in small beads, which were found along large stress fibers, along fine fibers, and in a poorly organized form near the lamellipodia. De novo assembly of beads was observed continuously within or near the lamellipodia, suggesting that myosin molecules may undergo a constant cycling between polymerized and unpolymerized states. The nascent structures then moved away from lamellipodia and became organized into linear arrays. Similar movement was also observed for beads already associated with linear structures, and may represent a continuous flux of myosin structures. The dynamic reorganization of myosin may play an important role in cell movement and polarity.     

Myosin II transport, organization, and phosphorylation: evidence for cortical flow/solation-contraction coupling during cytokinesis and cell locomotion.

The mechanism of cytokinesis has been difficult to define because of the short duration and the temporal-spatial dynamics involved in the formation, activation, force production, and disappearance of the cleavage furrow. We have investigated the structural and chemical dynamics of myosin II in living Swiss 3T3 cells from prometaphase through the separation and migration of daughter cells. The structural and chemical dynamics of myosin II have been defined using the semiautomated, multimode light microscope, together with a fluorescent analogue of myosin II and a fluorescent biosensor of myosin II regulatory light chain (RLC) phosphorylation at serine 19. The correlation of image data from live cells using different modes of light microscopy allowed interpretations not possible from single-mode investigations. Myosin II transported toward the equatorial plane from adjacent regions, forming three-dimensional fibers that spanned the volume of the equator during anaphase and telophase. A global phosphorylation of myosin II at serine 19 of the RLC was initiated at anaphase when cortical myosin II transport started. The phosphorylation of myosin II remained high near the equatorial plane through telophase and into cytokinesis, whereas the phosphorylation of myosin II at serine 19 of the RLC decreased at the poles. The timing and pattern of phosphorylation was the same as the shortening of myosin II-based fibers in the cleavage furrow. Myosin II-based fibers shortened and transported out of the cleavage furrow into the tails of the two daughter cells late in cytokinesis. The patterns of myosin II transport, phosphorylation, and shortening of fibers in the migrating daughter cells were similar to that previously defined for cells migrating in a wound in vitro. The temporal-spatial patterns and dynamics of myosin II transport, phosphorylation at serine 19 of the RLC, and the shortening and disappearance of myosin II-based fibers support the proposal that a combination of the cortical flow hypothesis and the solation-contraction coupling hypothesis explain key aspects of cytokinesis and polarized cell locomotion.     

Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.     

Visualization of myosin in living cells

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.     

Myosin phosphatase targeting subunit 1 affects cell migration by regulating myosin phosphorylation and actin assembly

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.     

Contraction and polymerization cooperate to assemble and close actomyosin rings around Xenopus oocyte wounds

Xenopus oocytes assemble an array of F-actin and myosin 2 around plasma membrane wounds. We analyzed this process in living oocytes using confocal time-lapse (four-dimensional) microscopy. Closure of wounds requires assembly and contraction of a classic "contractile ring" composed of F-actin and myosin 2. However, this ring works in concert with a 5-10-microm wide "zone" of localized actin and myosin 2 assembly. The zone forms before the ring and can be uncoupled from the ring by inhibition of cortical flow and contractility. However, contractility and the contractile ring are required for the stability and forward movement of the zone, as revealed by changes in zone dynamics after disruption of contractility and flow, or experimentally induced breakage of the contractile ring. We conclude that wound-induced contractile arrays are provided with their characteristic flexibility, speed, and strength by the combined input of two distinct components: a highly dynamic zone in which myosin 2 and actin preferentially assemble, and a stable contractile actomyosin ring.     

Laminin-311 (Laminin-6) fiber assembly by type I-like alveolar cells.

Two epithelial cell types cover the alveolar surface of the lung. Type II alveolar epithelial cells produce surfactant and, during development or following wounding, give rise to type I cells that are involved in gas exchange and alveolar fluid homeostasis. In culture, freshly isolated alveolar type II cells assume a more squamous (type I-like) appearance within 4 days after plating. They assemble numerous focal adhesions that associate with the actin cytoskeleton at the cell margins. These alveolar epithelial cells lose expression of type II cell markers including SP-C and after 4 days in culture express the type I cell marker T1alpha. Those cells that express T1alpha also deposit fibers of laminin-311 in their matrix. The latter appears to be related to their development of a type I phenotype because freshly isolated, primary type I cells also assemble laminin-311-rich fibers in vitro. A beta1 integrin antibody antagonist inhibits the assembly of laminin-311 matrix fibers. Moreover, the formation of laminin fibers is dependent on the activity of the small GTPases and is perturbed by ML-7, a myosin light chain kinase inhibitor. In summary, our data indicate that assembly of laminin-311 fibers by lung epithelial cells is integrin and actin cytoskeleton dependent, and that these fibers are characteristic of type I alveolar cells.     

Distribution of myosin mRNA during development and regeneration of skeletal muscle fibers

Myosin mRNA distribution among subcellular compartments of anterior tibialis muscles in rabbit is monitored by in situ hybridization. A high density of mRNA was widely distributed throughout myotubes from 29-day fetal muscle and from regenerating adult muscle. All cytoplasmic spaces contained mRNA except where scattered myofibrils and centrally located nuclei were found. In fibers from 22-week-old rabbits, myosin mRNA was concentrated under the sarcolemma and excluded from the consolidated myofibrils and peripheral nuclei. The dispersal of mRNA through the cytoplasm in myotubes suggests that translation of myosin is widespread and that rapid myofibril assembly can occur throughout the fiber.     

Relationship of Cytoskeletal Filaments to Annular Gap Junction Expression in Human Adrenal Cortical Tumor Cells in Culture

In addition to the well-characterized surface gap junctions expressed at contact sites between cells, annular gap junction profiles have been localized within the cytoplasm of some cell populations. To study and characterize these annular profiles, gap junction protein type was demonstrated with Western blot and immunocytochemistry. The distribution of annular gap junctions and the relationships to cytoskeletal elements were demonstrated with immunocytochemical, transmission electron microscopic, or image analysis with confocal microscopy techniques. SW-13 adrenal cortical tumor cells expressed α₁gap junctions at areas of cell to cell contact. In addition, α₁gap junction annular profiles were seen within the cytoplasm. Actin and myosin II were found closely associated with these annular gap junctions, while no physical association between tubulin- or vimentin-containing fibers and gap junction protein could be established. Disruption of microfilaments with cytochalasin B treatment (10 μg/ml, 1 h) resulted in a decrease in the average number and an increase in the average size of annular gap junctions compared to control populations. The results are consistent with a role for cytoskeletal elements containing actin and myosin II in annular gap junction turnover.     

Palmitoylation of apolipoprotein B is required for proper intracellular sorting and transport of cholesteroyl esters and triglycerides

Apolipoprotein B (apoB) is an essential component of chylomicrons, very low density lipoproteins, and low density lipoproteins. ApoB is a palmitoylated protein. To investigate the role of palmitoylation in lipoprotein function, a palmitoylation site was mapped to Cys-1085 and removed by mutagenesis. Secreted lipoprotein particles formed by nonpalmitoylated apoB were smaller and denser and failed to assemble a proper hydrophobic core. Indeed, the relative concentrations of nonpolar lipids were three to four times lower in lipoprotein particles containing mutant apoB compared with those containing wild-type apoB, whereas levels of polar lipids isolated from wild-type or mutant apoB lipoprotein particles appeared identical. Palmitoylation localized apoB to large vesicular structures corresponding to a subcompartment of the endoplasmic reticulum, where addition of neutral lipids was postulated to occur. In contrast, nonpalmitoylated apoB was concentrated in a dense perinuclear area corresponding to the Golgi compartment. The involvement of palmitoylation as a structural requirement for proper assembly of the hydrophobic core of the lipoprotein particle and its intracellular sorting represent novel roles for this posttranslational modification.     

M-RIP targets myosin phosphatase to stress fibers to regulate myosin light chain phosphorylation in vascular smooth muscle cells

Vascular smooth muscle cell contraction and relaxation are directly related to the phosphorylation state of the regulatory myosin light chain. Myosin light chains are dephosphorylated by myosin phosphatase, leading to vascular smooth muscle relaxation. Myosin phosphatase is localized not only at actin-myosin stress fibers where it dephosphorylates myosin light chains, but also in the cytoplasm and at the cell membrane. The mechanisms by which myosin phosphatase is targeted to these loci are incompletely understood. We recently identified myosin phosphatase-Rho interacting protein as a member of the myosin phosphatase complex that directly binds both the myosin binding subunit of myosin phosphatase and RhoA and is localized to actin-myosin stress fibers. We hypothesized that myosin phosphatase-Rho interacting protein targets myosin phosphatase to the contractile apparatus to dephosphorylate myosin light chains. We used RNA interference to silence the expression of myosin phosphatase-Rho interacting protein in human vascular smooth muscle cells. Myosin phosphatase-Rho interacting protein silencing reduced the localization of the myosin binding subunit to stress fibers. This reduction in stress fiber myosin phosphatase-Rho interacting protein and myosin binding subunit increased basal and lysophosphatidic acid-stimulated myosin light chain phosphorylation. Neither cellular myosin phosphatase, myosin light chain kinase, nor RhoA activities were changed by myosin phosphatase-Rho interacting protein silencing. Furthermore, myosin phosphatase-Rho interacting protein silencing resulted in marked phenotypic changes in vascular smooth muscle cells, including increased numbers of stress fibers, increased cell area, and reduced stress fiber inhibition in response to a Rho-kinase inhibitor. These data support the importance of myosin phosphatase-Rho interacting protein-dependent targeting of myosin phosphatase to stress fibers for regulating myosin light chain phosphorylation state and morphology in human vascular smooth muscle cells.