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At least 1,279 records · Page 71Linked to original sources
PubMed1999

An actin-binding protein of the Sla2/Huntingtin interacting protein 1 family is a novel component of clathrin-coated pits and vesicles.

The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an NH(2)-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled-coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R-green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.

3T3 Cells↗
PubMed2002

Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton.

Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead-cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin-catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.

Actins↗
PubMed2003

Oscillatory nucleocytoplasmic shuttling of the general stress response transcriptional activators Msn2 and Msn4 in Saccharomyces cerevisiae.

Msn2 and Msn4 are two related transcriptional activators that mediate a general response to stress in yeast Saccharomyces cerevisiae by eliciting the expression of specific sets of genes. In response to stress or nutritional limitation, Msn2 and Msn4 migrate from the cytoplasm to the nucleus. Using GFP-tagged constructs and high-resolution time-lapse video microscopy on single cells, we show that light emitted by the microscope also triggers this migration. Unexpectedly, the population of Msn2 or Msn4 molecules shuttles repetitively into and out of the nucleus with a periodicity of a few minutes. A large heterogeneity in the oscillatory response to stress is observed between individual cells. This periodic behavior, which can be induced by various types of stress, at intermediate stress levels, is not dependent upon protein synthesis and persists when the DNA-binding domain of Msn2 is removed. The cAMP-PKA pathway controls the sensitivity of the oscillatory nucleocytoplasmic shuttling. In the absence of PKA, Msn4 continues to oscillate while Msn2 is maintained in the nucleus. We show that a computational model based on the possibility that Msn2 and Msn4 participate in autoregulatory loops controlling their subcellular localization can account for the oscillatory behavior of the two transcription factors.

Binding Sites↗
PubMed2005

Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae.

Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance.

Cell Cycle↗
PubMed2006

GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells.

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin-Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

Alkaline Phosphatase↗
PubMed1982

Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation.

Thyone sperm were induced to undergo the acrosomal reaction with a calcium ionophore A23187 in sea water containing 50 mM excess CaCl2, and the extension of the acrosomal process was recorded with high-resolution, differential interference contrast video microscopy at 60 fields/sec. The length of the acrosomal process was measured at 0.25-s intervals on nine sperm. When the data were plotted as (length)2 vs. time, the points fell exactly on a straight line except for the initial and very final stages of elongation. Cytochalasin B alters the rate of elongation of the acrosomal process in a dose-dependent way, inhibiting the elongation completely at high concentrations (20 micrograms/ml). However, no inhibition was observed unless excess Ca++ was added to sea water. The concentration of actin in the periacrosomal cup of the unreacted sperm is as high as 160 mg/ml; we calculate this concentration from the number and lengths of the actin filaments in a fully reacted sperm, and the volume of the periacrosomal cup in the unreacted sperm. These results are consistent with the hypothesis proposed earlier that monomers add to the ends of the actin filaments situated at the tip of the growing acrosomal process (the preferred end for monomer addition), and that the rate of elongation of the process is limited by diffusion of monomers from the sperm head (periacrosomal cup) to the tip of the elongating process. During the extension of the acrosomal process, a few blebs distributed along its lengths move out with the process. These blebs maintain a constant distance from the tip of the growing process. At maximum length, the straight acrosomal process slackens into a bow, and numerous new blebs appear. A few seconds later, the process suddenly straightens out again and sometimes actually contracts. The behavior of the blebs indicates that membrane is inserted at the base of the growing acrosomal process, and that membrane assembly and water uptake must be coupled to actin assembly during elongation. We discuss how the dynamic balance of forces seems to determine the shape of the growing acrosomal process, and how actin assembly may be controlled during the acrosomal reaction.

Acrosome↗
PubMed1983

Rheological properties of living cytoplasm: endoplasm of Physarum plasmodium.

Magnetic sphere viscoelastometry, video microscopy, and the Kamiya double chamber method (Kamiya, N., 1940, Science [Wash. DC], 92:462-463.) have been combined in an optical and rheological investigation of the living endoplasm of Physarum polycephalum. The rheological properties examined were yield stress, viscosity (as a function of shear), and elasticity. These parameters were evaluated in directions perpendicular; (X) and parallel (Y) to the plasmodial vein. Known magnetic forces were used for measurements in the X direction, while the falling ball technique was used in the Y direction (Cygan, D.A., and B. Caswell, 1971, Trans. Soc. Rheol. 15:663-683; MacLean-Fletcher, S.D., and T.D. Pollard, 1980, J. Cell Biol., 85:414-428). Approximate yield stresses were calculated in the X and Y directions of 0.58 and 1.05 dyn/cm2, respectively. Apparent viscosities measured in the two directions (eta x and eta y) were found to fluctuate with time. The fluctuations in eta x and eta y were shown, statistically, to occur independently of each other. Frequency correlation with dynamoplasmograms indicated that these fluctuations probably occur independently of the streaming cycle. Viscosity was found to be a complex function of shear, indicating that the endoplasm is non-Newtonian. Plots of shear stress vs. rate of shear both parallel and perpendicular to the vein, showed that endoplasm is not a shear thinning material. These experiments have shown that living endoplasm of Physarum is an anisotropic viscoelastic fluid with a yield stress. The endoplasm appears not to be a homogeneous material, but to be composed of heterogeneous domains.

Cytoplasm↗
PubMed1994

Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes.

Light microscopic studies of phagocytosis showed that Salmonella typhimurium entered mouse macrophages enclosed in spacious phagosomes (SP). Viewed by time-lapse video microscopy, bone marrow-derived macrophages exposed to S. typhimurium displayed generalized plasma membrane ruffling and macropinocytosis. Phagosomes containing Salmonella were morphologically indistinguishable from macropinosomes. SP formation was observed after several methods of bacterial opsonization, although bacteria opsonized with specific IgG appeared initially in small phagosomes that later enlarged. In contrast to macropinosomes induced by growth factors, which shrink completely within 15 min, SP persisted in the cytoplasm, enlarging often by fusion with macropinosomes or other SP. A Salmonella strain containing a constitutive mutation in the phoP virulence regulatory locus (PhoPc) induced significantly fewer SP. Similar to Yersinia enterocolitica, PhoPc bacteria entered macrophages in close-fitting phagosomes, consistent with that expected for conventional receptor-mediated phagocytosis. These results suggest that formation of SP contributes to Salmonella survival and virulence.

Animals↗
PubMed1999

Dynamic interactions of macrophages with T cells during antigen presentation.

We have established a method for real-time video analysis of the interaction of antigen-presenting cells (APCs) with T cells. Green fluorescent protein expression controlled by a nuclear factor of activated T cells (NFAT)-responsive promoter permits the visualization of productive antigen presentation in single T cells. The readout is rapid (within 2 h) and semiquantitative and allows analysis by video microscopy and flow cytometry. Using this approach, we demonstrate that macrophages have the capacity to simultaneously activate multiple T cells. In addition, the interaction of T cells with macrophages is extraordinarily dynamic: after initial stable contact, the T cells migrate continuously on the surface of the macrophage and from APC to APC during productive antigen presentation. Thus, T cells sum up signals from multiple interactions with macrophages during stimulation.

Animals↗
PubMed2005

The adaptor molecules LAT and SLP-76 are specifically targeted by Yersinia to inhibit T cell activation.

T cell responses are critical to the survival of Yersinia-infected animals. Yersinia have the ability to directly suppress T lymphocyte activation through the virulence factor YopH, a tyrosine phosphatase. Using single cell video microscopy and FACS analysis, here we show that even an average of one Yersinia per T cell is sufficient to inhibit or alter T cell responses. This efficient inhibition is traced to specific targeting by YopH of the adaptor proteins, linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kD (SLP-76), which are crucial for T cell antigen receptor (TCR) signaling. A catalytically inactive YopH translocated via the type III secretory pathway from the bacteria into T cells primarily binds to LAT and SLP-76. Furthermore, among the proteins of the TCR signaling pathway, the tyrosine phosphorylation levels of LAT and SLP-76 are the most affected in T cells exposed to low numbers of Yersinia pseudotuberculosis. This is the first example showing that a pathogen targets these adaptor proteins in the TCR signaling pathway, suggesting a novel mechanism by which pathogens may efficiently alter T cell-mediated immune responses.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Modulation of rabbit ventricular cell volume and Na+/K+/2Cl- cotransport by cGMP and atrial natriuretic factor.

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2Cl- cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volume(test)/volume(control)) were determined. Exposure to 10 microM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 was 0.77 +/- 0.33 microM. Activating guanylate cyclase with 100 microM sodium nitroprusside also decreased cell volume to 0.889 +/- 0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 microM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2Cl- cotransport was tested by blocking the cotransporter with 10 microM bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 microM 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2Cl-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 microM 8-Br-cGMP prevented the effect of 1 microM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 microM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100 microM) amplified the volume decrease caused by a low dose of ANF (0.01 microM) approximately fivefold. In contrast, neither 100 microM 8-Br-cAMP nor 50 microM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also appear to modulate cell volume.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Swelling-activated Gd3+-sensitive cation current and cell volume regulation in rabbit ventricular myocytes.

The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6-0.9x normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (ICir,swell) that was reversibly blocked by Gd3+, but ICir,swell was not detected in isotonic or hypertonic media. This current was not related to IK1 because it was insensitive to Ba2+. The PK/PNa ratio for ICir,swell was 5.9 +/- 0.3, implying that inward current is largely Na+ under physiological conditions. Increasing bath K+ increased gCir,swell but decreased rectification. Gd3+ block was fitted with a K0.5 of 1.7 +/- 0.3 microM and Hill coefficient, n, of 1.7 +/- 0.4. Exposure to Gd3+ also reduced hypotonic swelling by up to approximately 30%, and block of current preceded the volume change by approximately 1 min. Gd3+-induced cell shrinkage was proportional to ICir,swell when ICir,swell was varied by graded swelling or Gd3+ concentration and was voltage dependent, reflecting the voltage dependence of ICir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that ICir,swell was sufficient to explain the majority of the volume change at -80 mV. In addition, swelling activated an outwardly rectifying Cl- current, ICl,swell. This current was absent after Cl- replacement, reversed at ECl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of ICl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, ICir,swell is expected to cause diastolic depolarization. Activation of ICir, swell also is likely to affect contraction and other physiological processes in myocytes.

Animals↗
PubMed1999

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL.

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Algorithms↗
PubMed1991

Initial stages of HIV-1 envelope glycoprotein-mediated cell fusion monitored by a new assay based on redistribution of fluorescent dyes.

Membrane fusion is an essential step in the infection of permissive cells with human immunodeficiency virus (HIV). Infected cells frequently fuse with each other, and then progress to form multinucleated giant cells (syncytia). To gain insight into mechanisms of HIV env-mediated membrane fusion, we developed a new assay for studying the initial events. The assay is based on the redistribution of fluorescent markers between membranes and cytoplasm of adjacent cells examined by means of fluorescence video microscopy. Membrane fusion between HIV-1 envelope glycoprotein (gp120/41) expressing effector cells and CD4+ target cells was observed 90 min after the association of cells, whereas the first syncytia only became apparent after 5 h. Moreover, membrane fusion events were observed under conditions where no syncytia were detected, for example, when the effector:target cell ratio was greater than 100:1, or less than 1:100. A significant number of cells with fused membranes were not involved in the syncytia. In order to determine whether quantitative differences in receptor expression might influence the extent of membrane fusion, we used laboratory-selected variants of CEM cells that differ in their expression of CD4. We found that CD4 is required on the target membrane for HIV env-mediated membrane fusion, but its extent is only partially dependent on CD4 surface concentration. The ability of those CEM variants to take part in HIV env-mediated membrane fusion did not correlate with their capacity to form syncytia. These findings indicate that additional steps are needed to form syncytia after membrane fusion.

CD4 Antigens↗
PubMed1992

LFA-1 adhesion molecules are not involved in the early stages of HIV-1 env-mediated cell membrane fusion.

A recently developed sensitive assay to examine the early stages of HIV-1 env-mediated cell fusion is based on the redistribution of fluorescent dyes between membranes and cytoplasm of adjacent cells, monitored by fluorescence video microscopy. This assay demonstrated that membrane fusion can occur under conditions where no syncytia are formed. Fusion started earlier than syncytia formation and was not very sensitive to HIV-1 env+/CD4+ cell ratios. In the current study, this assay was used to determine the role of LFA-1 in HIV-1 env-mediated membrane fusion and syncytia formation. CD4- LFA-1- Epstein-Barr virus transformed lines from two leukocyte adhesion deficiency patients were infected with recombinant vaccinia expressing gp120/41 (HIV-IIIB), and cocultured with CD4+ subclones of the human T cell line CEM, which were generated by chemical mutagenesis and express either normal (LFA-1+), or low levels of LFA-1 (LFA-1lo). It was found that the LFA-1lo T-cell clone formed much smaller and fewer syncytia compared to the LFA-1+ subclones, but both clones fused equally well with the gp120/41 expressing LFA-1- B cells as monitored by redistribution of fluorescent dyes. Furthermore, monoclonal antibodies against the LFA-1 molecules reduced the number of syncytia formed but had no effect on membrane fusion. These findings demonstrate that the adhesion molecule LFA-1 does not play a crucial role in the early events of HIV-1 env-mediated cell membrane fusion, but may contribute to the later events leading to giant cell formation.

Antibodies, Monoclonal↗
PubMed2000

Hyaluronic acid in cultured ovine tracheal cells and its effect on ciliary beat frequency in vitro.

Hyaluronic acid (hyaluronan, or HA) is secreted by submucosal glands, but its function in airway secretions other than influencing the rheology of mucus is not fully understood. HA is known to modulate cell behavior and to enhance sperm motility. Because sperm tails and cilia have the same microtubular structure, we studied the effect of HA on ciliary beat frequency (CBF) in vitro. CBF of cultured ovine airway epithelial cells was measured continuously by digital video microscopy. After removal of endogenous HA by hyaluronidase, cells were exposed to 50 to 100 microg/mL of HA at different times in culture. No change in CBF in response to HA was seen in cells cultured less than 7 days. After 7 days, however, 6 of 10 measured cells (from three different sheep) showed a transient CBF increase from a baseline of 6.4 +/- 0.3 Hz (mean +/- SE) to 7.4 +/- 0.4 Hz or 16% above baseline (p < 0.05). At these time points (but not before), cytochemical staining was positive for endogenous HA using a biotinylated HA-binding protein. These data suggest that HA can increase CBF of tracheal epithelial cells only late in culture when HA is able to bind to an unspecified cell surface structure. Because this binding has a physiological effect, we hypothesize that it is an HA-binding receptor, that is either transiently expressed late in culture or initially destroyed by the protease treatment for cell dispersion.

Animals↗
PubMed1999

Taxol suppresses dynamics of individual microtubules in living human tumor cells.

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30-100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

Adenocarcinoma↗
PubMed2000

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration.

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.

Actins↗