Gravity-induced reorientation of cortical microtubules observed in vivo

Cortical microtubules play an important role during morphogenesis by determining the direction of cellulose deposition. Although many triggers are known that can induce the reorientation of cortical plant microtubules, the reorientation mechanism has remained obscure. In our approach, we used gravitropic stimulation which is a strong trigger for microtubule reorientation in epidermal cells of maize coleoptiles. To visualize the gravitropically induced microtubule reorientation in living cells, we injected rhodamine-conjugated tubulin into epidermal cells of intact maize coleoptiles that were exposed to gravitropic stimulation. From these in vivo observations, we propose a reorientation mechanism consisting of four different stages: (1) a transitional stage with randomly organized microtubules; (2) emergence of a few microtubules in a slightly oblique orientation; (3) co-alignment: neighbouring microtubules adopt the oblique orientation resulting in parallel organized microtubules; and (4) the angle of these parallel, organized microtubules increases gradually. Thus, the overall reorientation process could include selective stabilization/ disassembly of microtubules (stage 2) as well as movement of individual microtubules (stages 3 and 4).     

The cyclic reorientation of cortical microtubules on walls with a crossed polylamellate structure: effects of plant hormones and an inhibitor of protein kinases on the progression of the cycle

Summary The outer tangential wall (OTW) of epidermal cells of azuki bean epicotyls has a crossed polylamellate structure, in which lamellae of longitudinal cellulose microfibrils alternate with lamellae of transverse cellulose microfibrils. This implies that the cyclic reorientation of cortical microtubules (MTs) from longitudinal to transverse and from transverse to longitudinal occurs on the OTW. Treatment with a solution that contained no auxin caused the accumulation of cells with longitudinal MTs, suggesting that auxin is required for the reorientation of MTs from longitudinal to transverse during the reorientation cycle. Treatment with 6-dimethylaminopurine (DMAP), an inhibitor of protein kinases that promoted the reorientation of MTs from transverse to longitudinal, resulted in the accumulation of cells with longitudinal MTs. Subsequent treatment with auxin caused a marked increase in the percentage of cells with transverse MTs and then a decrease in the percentage, indicating that the reorientation of MTs from longitudinal to transverse and then from transverse to longitudinal occurred during treatment with auxin. The percentage of cells with transverse MTs decreased more slowly in segments that had been pretreated with gibberellin A₃ (GA) than in segments that had been pretreated without GA, suggesting that GA, in cooperation with auxin, caused the suppression of the reorientation of MTs from transverse to longitudinal.Abbreviations BL brassinolide - BSA bovine serum albumin - GA gibberellin A₃ - DMAP 6-dimethylaminopurine - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IAA indoleacetic acid - MT microtubule - OTW outer tangential wall - PBS phosphate-buffered saline Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Europium III binding and the reorientation of magnetically aligned bicelles: insights from deuterium NMR spectroscopy.

Solid-state deuterium ((2)H) NMR spectroscopy was used to study the reorientation of magnetically ordered bicelles in the presence of the paramagnetic lanthanide Eu(3+). Bicelles were composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) plus 1,2-dihexanoyl-sn-glycero-3-phosphocholine plus either the anionic lipid 1,2-dimyristoyl-sn-3-phosphoglycerol, or the cationic lipid 1,2-dimyristoyl-3-trimethyl ammonium propane. Alignment of the bicelles in the magnetic field produced (2)H NMR spectra consisting of a pair of quadrupole doublets, one from the alpha-deuterons and one from the beta-deuterons of DMPC-alpha,beta-d(4). Eu(3+) addition induced the appearance of a second set of quadrupole doublets, having approximately twice the quadrupolar splittings of the originals, and growing progressively in intensity with increasing Eu(3+), at the expense of the intensity of the originals. The new resonances were attributed to bicelles having a parallel alignment with respect to the magnetic field, as opposed to the perpendicular alignment preferred in the absence of Eu(3+). Therefore, the equilibrium degree and kinetics of reorientation could be evaluated from the (2)H NMR spectra. For more cationic initial surface charges, higher amounts of added Eu(3+) were required to induce a given degree of reorientation. However, the equilibrium degree of bicellar reorientation was found to depend solely on the amount of bound Eu(3+), regardless of the bicelle composition. The kinetics of reorientation were a function of lipid concentration. At high lipid concentration, a single fast rate of reorientation (minutes) described the approach to the equilibrium degree of orientation. At lower lipid concentrations, two rates processes were discernible: one fast (minutes) and one slow (hours). The data indicate, therefore, that bicelle reorientation is a phase transition made critical by bicelle-bicelle interactions.     

Interrogating the role of liposome size in mediating the dynamics of a chromophore in the acyl chain region of a phospholipid bilayer

We have examined the temperature-dependent reorientation dynamics of perylene imbedded in bilayers of 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), where the bilayers exist in the form of unilamellar vesicles. Previous work using 100-nm diameter DMPC vesicles has shown that the phase transition from the gel phase to the fluid phase can be detected using the reorientation dynamics of perylene. In this work we explore the vesicle size dependence of the perylene reorientation dynamics in DMPC vesicles. The size of the vesicles is determined by extrusion and the reorientation dynamics of perylene are measured as a function of vesicle size between 100-nm and 5-microm diameter. We find that, while the phase transition for DMPC is seen in smaller vesicles, perylene becomes insensitive to the phase transition for vesicles larger than ca. 800-nm diameter. We also find a discontinuous change in perylene reorientation dynamics with increasing vesicle size, and we consider this result in the context of the location of perylene within the bilayer.     

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.     

Degeneration affects the fiber reorientation of human annulus fibrosus under tensile load

The angled, lamellar structure of the annulus fibrosus is integral to its load-bearing function. Reorientation of this fiber structure with applied load may contribute to nonlinear mechanical behavior and to large increases in tensile modulus. Fiber reorientation has not yet been quantified for loaded non-degenerated and degenerated annulus fibrosus tissue. The objective of this study was to measure fiber reorientation and mechanical properties (toe- and linear-region modulus, transition strain, and Poisson's ratio) of loaded outer annulus fibrosus tissue using a new application of FFT image processing techniques. This method was validated for quantification of annulus fiber reorientation during loading in this study. We hypothesized that annulus fibrosus fibers would reorient under circumferential tensile load, and that fiber reorientation would be affine. Additionally, we hypothesized that degeneration would affect fiber reorientation, toe-region modulus and Poisson's ratio. Annulus fibrosus fibers were found to reorient toward the loading direction, and degeneration significantly decreased fiber reorientation (the fiber reorientation parameter, m(FFT)=-1.70 degrees /% strain for non-degenerated and -0.95 degrees /% strain for degenerated tissue). Toe-region modulus was significantly correlated with age (r=0.6). Paired t-tests showed no significant difference in the fiber reorientation parameter calculated experimentally with that calculated using an affine prediction. Thus, an affine prediction is a good approximation of fiber reorientation. The findings of this study add to the understanding of overall disc mechanical behavior and degeneration.     

Nox4-dependent activation of cofilin mediates VSMC reorientation in response to cyclic stretching

Vascular smooth muscle cells (VSMCs) are subjected to various types of mechanical forces within the vessel wall. Although it is known that VSMCs undergo cell body reorientation in response to mechanical stimulation, how this mechanical stretch is transduced within the cell into biochemical signals causing cytoskeleton reorganization remains unclear. Cofilin, a protein that controls actin dynamics, is activated by Slingshot phosphatase-dependent serine 3 dephosphorylation by redox-dependent mechanisms. Nox4 is a main source of reactive oxygen species (ROS) in the vessel wall that localizes in association with the cytoskeleton. Therefore, we hypothesize that Nox4 mediates redox-dependent activation of cofilin, which is required for cytoskeletal reorganization and cell reorientation after mechanical stimulation. In this study, we found that mechanical stretch stimulates ROS production in VSMCs and that the signaling that leads to cell reorientation requires hydrogen peroxide but not superoxide. Indeed, mechanical stretch induces cofilin activation and stretch-induced cytoskeletal reorganization, and cell reorientation is inhibited in cells where cofilin activity has been downregulated. Importantly, Nox4-deficient cells fail to activate cofilin and to undergo cell reorientation, a phenotype rescued by the expression of a constitutively active cofilin mutant. Our results demonstrate that in VSMCs mechanical stimulation activates cofilin by a Nox4-dependent mechanism and that this pathway is required for cytoskeleton reorganization and cell reorientation.     

Proposed role of microfilaments in the cell reorientation that accompanies somite formation in Xenopus

Somite formation involves a reorientation of the cells of the paraxial mesoderm but the underlying mechanism of this movement has never been demonstrated. The present investigation shows bundles of myofibril-like material in the ventrocranial corners of some of the paraxial mesoderm cells of Xenopus prior to reorientation and consequent somite formation.     

Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament-disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts.     

Ethylene-induced microtubule reorientation is essential for fast inhibition of root elongation in Arabidopsis

Microtubule reorientation is a long-standing observation that has been implicated in regulating the inhibitory effect of ethylene on axial elongation of plant cells. However, the signaling mechanism underlying ethylene-induced microtubule reorientation has remained elusive. Here, we reveal, by live confocal imaging and kinetic root elongation assays, that the time courses of ethylene-induced microtubule reorientation and root elongation inhibition are highly correlated, and that microtubule reorientation is required for the full responsiveness of root elongation to ethylene treatment. Our genetic analysis demonstrated that the effect of ethylene on microtubule orientation and root elongation is mainly transduced through the canonical linear ethylene signaling pathway. By using pharmacological and genetic analyses, we demonstrate further that the TIR1/AFBs-Aux/IAAs-ARFs auxin signaling pathway, but not the ABP1-ROP6-RIC1 auxin signaling branch, is essential for ethylene-induced microtubule reorientation and root elongation inhibition. Together, these findings offer evidence for the functional significance and elucidate the signaling mechanism for ethylene-induced microtubule reorientation in fast root elongation inhibition in Arabidopsis.     

Interaction between Green and Far-Red Light on the Low Fluence Rate Chloroplast Orientation in Mougeotia

Continuous irradiation of Mougeotia with linearly polarized green light (550 nanometers, 0.2 watt per square meter) induces a change in the orientation of its chloroplast from profile to face position, if the electrical vector of the green light is vibrating normal to the cell axis. This change is complete within 25 minutes of the onset of irradiation. In contrast, if the electrical vector of the green light is parallel to the cell axis, no chloroplast reorientation is induced, even with a fluence rate as high as 3 watts per square meter. Furthermore, unpolarized far-red light (727 nanometers, 2 watts per square meter) given alone has no effect on chloroplast reorientation. Simultaneous and continuous irradiation with polarized green light, regardless of its plane of polarization, together with unpolarized far-red light, however, does lead to chloroplast reorientation. These data indicate that, in addition to the red-absorbing form of phytochrome, there exists in Mougeotia another sensory pigment absorbing green light.     

Reorientation of the acetyl group of the photoactive bacteriopheophytin in reaction centers of Rhodobacter sphaeroides: an ENDOR/TRIPLE resonance study

The freeze-trapped bacteriopheophytin alpha radical anion phi(*)A- has been investigated by 1H-ENDOR/Special TRIPLE resonance spectroscopy in photosynthetic reaction centers of Rhodobacter sphaeroides, in which the Tyr at position M210 had been replaced by either Phe, Leu, His or Trp. In the wild type reaction center and the mutants YF(M210) and YW(M210) two distinct states of phi(*)A-, denoted I(*)1- and I(*)2-, can be stabilized below 200 K. The state I(*)1 is metastable and relaxes to I(*)2- as the temperature is raised from 135 K to 180 K. The difference in the electronic structure of phi(*)A- between the two states is interpreted in terms of a conformational change of phiA after freeze-trapping, involving a reorientation of the 3-acetyl group with respect to the macrocycle of the bacteriopheophytin. This interpretation is supported by the results of RHF-INDO/SP calculations. In the YH(M210) reaction center only one phiA- state is obtained that is distinct from I(*)1- and I(*)2, and the observed electronic structure indicates an almost in-plane orientation of the 3-acetyl group. This is consistent with the proposal that a hydrogen bond is formed between His M210 and the 3(1)-keto oxygen of phiA that impedes the reorientation of the acetyl group. Only one phi(*)A- state is observed in the YL(M210) reaction center, which is similar to the metastable state I(*)1 in the wild type complex. This result is interpreted in terms of a steric hindrance of the reorientation of the 3-acetyl group that is exerted by the side chain of Leu at position M210. Possible implications of these findings for the mechanism of electron transfer in bacterial reaction centers are discussed.     

MTOC reorientation occurs during FcgammaR-mediated phagocytosis in macrophages

Cell polarization is essential for targeting signaling elements and organelles to active plasma membrane regions. In a few specialized cell types, cell polarity is enhanced by reorientation of the MTOC and associated organelles toward dynamic membrane sites. Phagocytosis is a highly polarized process whereby particles >0.5 microm are internalized at stimulated regions on the cell surface of macrophages. Here we provide detailed evidence that the MTOC reorients toward the site of particle internalization during phagocytosis. We visualized MTOC proximity to IgG-sRBCs in fixed RAW264.7 cells, during live cell imaging using fluorescent chimeras to label the MTOC and using frustrated phagocytosis assays. MTOC reorientation in macrophages is initiated by FcgammaR ligation and is complete within 1 h. Polarization of the MTOC toward the phagosome requires the MT cytoskeleton and dynein motor activity. cdc42, PI3K, and mPAR-6 are all important signaling molecules for MTOC reorientation during phagocytosis. MTOC reorientation was not essential for particle internalization or phagolysosome formation. However Golgi reorientation in concert with MTOC reorientation during phagocytosis implicates MTOC reorientation in antigen processing events in macrophages.     

Effects of cyclophosphamide on the secondary palate development in golden Syrian hamster: teratology, morphology, and morphometry

Cyclophosphamide (CP), when injected in hamster mother between days 9 and 11 of pregnancy, was teratogenic in fetuses. On the basis of a morphological study it was deduced that CP delayed the reorientation of hamster palatal shelves by 16-20 h. In a subsequent experiment, in both control and CP-treated palatal shelves, the numbers of epithelial and mesenchymal cells were counted and cross-sectional area was measured. DNA synthesis, measured by 3H-thymidine incorporation, was used as an index of growth by cell proliferation. The results showed that during the vertical development of palatal shelves, the mesenchymal cells reached their peak number during the initial 24 hours, i.e., at the end of the second peak in DNA synthesis, and remained unchanged thereafter throughout reorientation. The shelf area also showed rapid increase during the initial 24 h followed by a spurt 2 h prior to reorientation. Cyclophosphamide prolonged the acquisition of these features by affecting the mesenchymal cells and consequently delayed the reorientation of the vertical shelves until such time that the number of healthy mesenchymal cells and shelf area were restored to the control values. The data lend further support to the hypothesis that the acquisition of a specific number of cells and shelf volume, during vertical palatal development, may be essential for palatal shelf reorientation.     

High resolution 1H nuclear magnetic resonance of a transmembrane peptide.

Although the strong 1H-1H dipolar interaction is known to result in severe homogeneous broadening of the 1H nuclear magnetic resonance (NMR) spectra of ordered systems, in the fluid phase of biological and model membranes the rapid, axially symmetric reorientation of the molecules about the local bilayer normal projects the dipolar interaction onto the motional symmetry axis. Because the linewidth then scales as (3 cos2 theta-1)/2, where theta is the angle between the local bilayer normal and the magnetic field, the dipolar broadening has been reduced to an "inhomogeneous" broadening by the rapid axial reorientation. It is then possible to obtain high resolution 1H-NMR spectra of membrane components by using magic angle spinning (MAS). Although the rapid axial reorientation effectively eliminates the homogeneous dipolar broadening, including that due to n = 0 rotational resonances, the linewidths observed in both lipids and peptides are dominated by low frequency motions. For small peptides the most likely slow motions are either a "wobble" or reorientation of the molecular diffusion axis relative to the local bilayer normal, or the reorientation of the local bilayer normal itself through surface undulations or lateral diffusion over the curved surface. These motions render the peptide 1H-NMR lines too broad to be observed at low spinning speeds. However, the linewidths due to these slow motions are very sensitive to spinning rate, so that at higher speeds the lines become readily visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-linking of SH-groups in the erythrocyte membrane enhances transbilayer reorientation of phospholipids. Evidence for a limited access of phospholipids to the reorientation sites

Oxidation of erythrocyte membrane SH-groups and concomitant cross-linking of spectrin, which induce a partial loss of phospholipid asymmetry (Haest, C.W.M., Plasa, G., Kamp, D. and Deuticke, B. (1978) Biochim. Biophys. Acta 509, 21-32) are now shown to result in a remarkable increase of the rates of transbilayer reorientation of exogenously incorporated lysophospholipids. Reorientation of both, neutral lysophosphatidylcholine and of negatively charged lysophosphatidylserine is enhanced. A decrease of the activation energy of the reorientation process as well as quantitative changes of the dependence of reorientation on the lysophosphatidylcholine and cholesterol content of the membrane indicate formation of new reorientation sites or modification of existing sites. A common mechanism may underly the formation of reorientation sites and the occurrence of leaks for small solutes (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210) subsequent to oxidation of membrane SH-groups. Whereas exogenous lysophospholipids completely equilibrate between the two lipid layers regardless of the extent of oxidation of SH-groups, endogenous inner layer phospholipids become available for reorientation in a graded way. Native phospholipid asymmetry is therefore not the result of a low transbilayer mobility of phospholipids, but probably due to a lack of access of inner layer phospholipids to the reorientation sites.     

Computer-assisted analysis of hyaluronate distribution during morphogenesis of the mouse secondary palate

Hyaluronate mediated extracellular matrix swelling has been hypothesized to play a major role in reorientation of the secondary palatal shelves. A computer-assisted method utilizing image registration and subtraction was used to visualize the distribution of hyaluronate (HA) during morphogenesis of the secondary palate. Patterns of HA distribution in anterior, posterior and presumptive soft palate were examined in the secondary palatal shelves of CD-1 mouse fetuses that were 30, 24 and 18 h prior to, and at the time of, shelf reorientation. Adjacent serial sections were taken from each shelf region of three to six specimens from a minimum of three litters for each gestational age. One section was incubated in buffer as a control, the other digested with Streptomyces hyaluronidase to specifically remove HA. Both sections were stained with Alcian blue to visualize the extracellular matrix and counterstained with nuclear fast red to visualize cells. Two different videoimages were then digitized for each tissue section, one using wavelengths of light that were at or near the maximum absorbance of the matrix stain, the other using wavelengths that were at the maximum absorbance of the cellular stain. Thus, a matrix image and a cell image of both control and digested sections were produced. Next, the cell image was subtracted from its respective matrix image, resulting in a control matrix-only image and a digested (HA-removed) matrix-only image. These images were mathematically warped to one another, if necessary, and registered with one another. The digested image was then subtracted from the control image. The resultant difference picture displayed the pattern and relative intensities of HA distribution across the tissue section. Prior to and during shelf reorientation, unique region-specific patterns of HA distribution and relative intensity were identified which became homogeneous after reorientation. Presumptive soft palate shows the most extensive and intense patterns of HA distribution, followed by the posterior region. The anterior region has the most sparse pattern of the three regions examined. The results are consistent with the hypothesized role of HA in shelf reorientation.     

Role of 1,4,5-inositol triphosphate-induced Ca2+ release in pollen tube orientation

 Pollen tube reorientation is a dynamic cellular event crucial for successful fertilization. Previously, it was shown that reorientation is preceded by an asymmetric increase of cytosolic free calcium ([Ca²⁺]_c) in the side of the apex to which the cell will bend. In order to find the targets for this signal transduction pathway, the effects of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in the reorientation process were analyzed. Ins(1,4,5)P₃ was artificially increased in different cell domains by localized photoactivation of caged Ins(1,4,5)P₃ and its effects on [Ca²⁺]_c monitored by ion confocal microscopy. It was found that photolysis of caged Ins(1,4,5)P₃ in the nuclear or subapical region resulted in a transient increase in [Ca²⁺]_c and reorientation of the growth axis, while photolysis in the apex frequently resulted in disturbed growth or tip bursting. Perfusion of the cells with the Ins(1,4,5)P₃ receptor blocker heparin prior to photoactivation inhibited the increase in [Ca²⁺]_c and no reorientation was observed. Ca²⁺ release from Ins(1,4,5)P₃-dependent stores localized in the shank of the tube thus seems to be part of the signal transduction pathway that controls tube guidance, although not the primary stimulus leading to reorientation. Received: 5 May 1998 / Accepted: 11 June 1998     

A Morphometric Analysis of the Redistribution of Organelles in Columella Cells of Horizontally-oriented Roots of Zea mays

In order to determine what structural changes in graviperceptivecells are associated with the onset of root gravicurvature,the redistribution of organelles in columella cells of horizontally-oriented,graviresponding roots of Zea mays has been quantified. Rootgravicurvature began by 15 min after reorientation, and didnot involve significant changes in the (i) volume of individualcolumella cells or amyloplasts, (ii) relative volume of anycellular organelle, (iii) number of amyloplasts per columellacell, or (iv) surface area or cellular location of endoplasmicreticulum. Sedimentation of amyloplasts began within 1 to 2min after reorientation, and was characterized by an intenselystaining area of cytoplasm adjacent to the sedimenting amyloplasts.By 5 min after reorientation, amyloplasts were located in thelower distal corner of columella cells, and, by 15 min afterreorientation, overlaid the entire length of the lower cellwall. No consistent contact between amyloplasts and any cellularstructure was detected at any stage of gravicurvature. Centrally-locatednuclei initially migrated upward in columella cells of horizontally-orientedroots, after which they moved to the proximal ends of the cellsby 15 min after reorientation. No significant pattern of redistributionof vacuoles, mitochondra, dictyosomes, or hyaloplasm was detectedthat correlated with the onset of gravicurvature. These resultsindicate that amyloplasts and nuclei are the only organelieswhose movements correlate positively with the onset of gravicurvatureby primary roots of this cultivar of Zea mays. Zea mays, root gravitropism, ultrastructure, morphometry, graviperception     

The formin mDia regulates GSK3beta through novel PKCs to promote microtubule stabilization but not MTOC reorientation in migrating fibroblasts

In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3beta (GSK3beta) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3beta inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3beta in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3beta functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3beta phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3beta through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3beta Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3beta through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.