Defective Actin Research Articles

The Temperature-Sensitive Role of Cryptococcus neoformans ROM2 in Cell Morphogenesis

ROM2 is associated with Cryptococcus neoformans virulence. We examined additional roles of ROM2 in C. neoformans and found that ROM2 plays a role in several cell functions specifically at high temperature conditions. Morphologically rom2 mutant cells demonstrated a “tear”-like shape and clustered together. A sub-population of cells had a hyperelongated phenotype at restrictive growth conditions. Altered morphology was associated with defects in actin that was concentrated at the cell periphery and with abnormalities in microtubule organization. Interestingly, the ROM2 associated defects in cell morphology, location of nuclei, and actin and microtubule organization were not observed in cells grown at temperatures below 37°C. These results indicate that in C. neoformans, ROM2 is important at restrictive temperature conditions and is involved in several cell maintenance functions.

Rho GTPase Cdc42 coordinates hematopoietic stem cell quiescence and niche interaction in the bone marrow

Adult hematopoietic stem cells (HSCs) exist in a relatively quiescent state in the bone marrow (BM) microenvironment to fulfill long-term self-renewal and multilineage differentiation functions, an event that is tightly regulated by extrinsic and intrinsic cues. However, the mechanism coordinating the quiescent state of HSCs and their retention in the BM microenvironment remains poorly understood. In a conditional-knockout mouse model, we show that Cdc42(-/-) HSCs enter the active cell cycle, resulting in significantly increased number and frequency of the stem/progenitor cells in the BM. Cdc42 deficiency also causes impaired adhesion, homing, lodging, and retention of HSCs, leading to massive egress of HSCs from BM to distal organs and peripheral blood and to an engraftment failure. These effects are intrinsic to the HSCs and are associated with deregulated c-Myc, p21(Cip1), beta1-integrin, and N-cadherin expressions and defective actin organization. Thus, Cdc42 is a critical coordinator of HSC quiescence maintenance and interaction with the BM niche.

Association between CFL1gene polymorphisms and spina bifida risk in a California population

BackgroundCFL1 encodes human non-muscle cofilin (n-cofilin), which is an actin-depolymerizing factor and is essential in cytokinesis, endocytosis, and in the development of all embryonic tissues. Cfl1 knockout mice exhibit failure of neural tube closure at E10.5 and die in utero. We hypothesized that genetic variation within the human CFL1 gene may alter the protein's function and result in defective actin depolymerizing and cellular activity during neural tube closure. Such alterations may be associated with an increased risk for neural tube defects (NTDs).MethodsHaving re-sequenced the human CFL1 gene and identified five common single nucleotide polymorphisms (SNPs) in our target population, we investigated whether there existed a possible association between the genetic variations of the CFL1 gene and risk of spina bifida. Samples were obtained from a large population-based case-control study in California. Allele association, genotype association and haplotype association were evaluated in two different ethnicity groups, non-Hispanic white and Hispanic white.ResultsHomozygosity for the minor alleles of the SNPs studied (rs652021, rs665306, rs667555, rs4621 and rs11227332) appeared to produce an increased risk for spina bifida. Subjects with the haplotype composed of all minor alleles (CCGGT) appeared to have increased spina bifida risk (OR = 1.6, 95% CI: 0.9~2.9), however, this finding is not statistically significant likely due to limited sample size.ConclusionThe sequence variation of human CFL1 gene is a genetic modifier for spina bifida risk in this California population.

2.022 Cognitive and locomotor deficits accompanied by congophilic aggregate formation in the Drosophila model for defective actin remodeling

2.022 Cognitive and locomotor deficits accompanied by congophilic aggregate formation in the Drosophila model for defective actin remodeling

2.418 Cognitive and locomotor deficits accompanied by congophilic aggregate formation in the Drosophila model for defective actin remodeling

2.418 Cognitive and locomotor deficits accompanied by congophilic aggregate formation in the Drosophila model for defective actin remodeling

Loss of IRF-4–binding protein leads to the spontaneous development of systemic autoimmunity

IFN regulatory factor 4-binding (IRF-4-binding) protein (IBP) is a novel type of activator of Rho GTPases that is recruited to the immunological synapse upon TCR stimulation. Here we demonstrate that loss of IBP leads to the spontaneous development of a systemic autoimmune disorder characterized by the accumulation of effector/memory T cells and IgG+ B cells, profound hypergammaglobulinemia, and autoantibody production. Similar to human SLE, this syndrome primarily affects females. T cells from IBP-deficient mice are resistant to death in vitro as well as in vivo and exhibit selective defects in effector function. In the absence of IBP, T cells respond suboptimally to TCR engagement, as demonstrated by diminished ERK1/2 activation, decreased c-Fos induction, impaired immunological synapse formation, and defective actin polymerization. Transduction of IBP-deficient T cells with a WT IBP protein, but not with an IBP mutant lacking the Dbl-like domain required for Rho GTPase activation, rescues the cytoskeletal defects exhibited by these cells. Collectively, these findings indicate that IBP, a novel regulator of Rho GTPases, is required for optimal T cell effector function, lymphocyte homeostasis, and the prevention of systemic autoimmunity.

A new approach for actinic defect inspection of EUVL multilayer mask blanks: Standing wave photoemission electron microscopy

Extreme ultraviolet lithography (EUVL) at 13.5 nm is the next generation lithography technique capable of printing sub-50 nm structures. With decreasing feature sizes to be printed, the requirements for the lithography mask also become more stringent in terms of defect sizes and densities that are still acceptable and the development of lithography optics has to go along with the development of new mask defect inspection techniques that are fast and offer high resolution (preferable in the range of the minimum feature size) at the same time. We report on the development and experimental results of a new ‘at wavelength’ full-field imaging technique for defect inspection of multilayer mask blanks for EUV lithography. Our approach uses a photoemission electron microscope (PEEM) in a normal incidence illumination mode at 13 nm to image the photoelectron emission induced by the EUV wave field on the multilayer mask blank surface. We show that by these means, buried defects in the multilayer stack can be probed down to a lateral size of 50 nm, which was the smallest pre-defined structure size under study so far. The PEEMs spatial resolution has been proven to be as small as 29 nm edge slope width in measurements with 250 nm radiation. Therefore, the capability of this technique for actinic measurements at 13 nm radiation is anticipated to be well below 50 nm.

High-resolution actinic defect inspection for extreme ultraviolet lithography multilayer mask blanks by photoemission electron microscopy

We report on the development and first experimental results of a “at wavelength” full-field imaging technique for defect inspection of multilayer mask blanks for extreme ultraviolet (EUV) lithography. According to the International Semiconductor Roadmap by Sematech, less than 5×10−3 defects per cm2 should be present on such multilayer mask blank to enable mass production of microelectronics using EUV lithography, thus fast high-resolution methods for mask defect inspection and localization are needed. Our approach uses a photoemission electron microscope in a normal incidence illumination mode at 13 nm to image the photoelectron emission induced by the EUV wave field on the multilayer mask blank surface. We show that by these means, buried defects in the multilayer stack can be probed down to a lateral size of 50 nm.

A Critical Role of the Rho Family GTPase Cdc42 in Hematopoietic Stem Cell Mobilization, Homing, Engraftment and Differentiation.

The Rho family GTPase Cdc42 has emerged as a key signaling molecule with unique roles in multiple hematopoietic cell lineages. To investigate the physiologic role of Cdc42 in hematopoietic stem cells/progenitors (HSC/Ps), we bred conditional cdc42flox/floxmice with Mx-Cre mice that allowed interferon-inducible cdc42 deletion in hematopoietic cells in vivo following polyI:C induction. Loss of Cdc42 expression in the bone marrow caused significant leukocytosis with neutrophilia in peripheral blood (PB) (Table). Concurrently, deletion of cdc42 in the bone marrow led to a massive efflux of HSC/Ps from bone marrow to PB, liver and spleen (Table) and increased proliferative activity of HSC/Ps. Consequently, flow analysis further revealed that the number of Lin-Sca-1+c-Kit+ (LSK) HSCs in BM, PB, spleen and liver increased significantly following cdc42 deletion (Table). Transplantation of the cdc42−/− bone marrow cells into NOD/SCID recipient mice or competitive transplantation into BoyJ recipient mice showed that cdc42 deficiency in the bone marrow caused the impaired engraftment (Table) and the failure of hematopoiesis after a transient increase of cell cycle rate of HSCs. The engraftment defect is associated with a homing deficiency (Table). The cdc42−/− (Lin−c-Kit+) HSC/Ps showed defects in cortical F-actin assembly, adhesion to fibronectin and directional migration in response to SDF-1α (Table). These data suggest that defective actin structure and adhesion of the cdc42−/− HSC/Ps may contribute to the loss of retention of the cells in the BM niche and to the mobilization phenotype. Moreover, deletion of cdc42 resulted in significantly reduced thymus cellularity, T cell, B cell population, and decreased Ter119+ cell number associated with markedly decreased CFU-E activity of the bone marrow cells (Table). Two additional complimentary approaches, BM-reconstitution by cdc42−/− HSCs and reciprocal (rescue) transplantation by WT HSCs, further demonstrated that the Cdc42 loss-of-function phenotypes are hematopoietic cell intrinsic. Taken together these results implicate Cdc42 as a critical regulator of HSC mobilization, homing and engraftment, and indicate that Cdc42 is involved in erythropoiesis and in T- and B-cell differentiation and homeostasis.WTCdc42−/−White blood cell count, × 109 /L11.0±2.540.8±3.9†Neutrophil count, × 109 /L4.6±0.834.1±4.7†CFU-C per 105 liver cells0.13±0.12169±132*CFU-C per 104 spleen cells1.36±1.0568.4±55.6**CFU-C per 100μl PB3.67±258.8±47.3**LSK HSCs in BM0.52%±0.22%0.94%±0.45%*LSK HSCs in PB0.13%±0.08%0.31%±0.15%*LSK HSCs in spleen0.37%±0.20%0.63%±0.23%*LSK HSCs in liver0.23%±0.10%0.70%±0.18%*Engraftment in NOD/SCID mice35.0%±4.9%23.0%±7.1%*Competitive transplantation61.5%±6.7%22.2%±2.9%**CFU-C Homing to BM (% injected)0.85%± 0.11%0.28%± 0.12%**Adhesion to fibronectin (%plating)50.8%±2.5%28.8%±4.0%**Migration to SDF (%input)26.2%±5.3%5.4%±0.9%†CD3+ T cells in BM5.4%±0.22%2.7%±0.45%**B220+ B cells in BM8.4%±4.6%1.5%±2.0%†Ter119+ cells in BM15.2%±9.3%6.0%±4.2%*CFU-E per 105 BM cells87.8±9.725.6±4.2†*p<0.05**p<0.005†p<0.001

The Role of SPECs, Small Cdc42-binding Proteins, in F-actin Accumulation at the Immunological Synapse

SPEC1 and SPEC2 are structurally similar Cdc42-binding proteins of 79 and 84 amino acid residues, respectively. We investigated the role of SPEC2 in T cell function due to its high mRNA expression in lymphocytes. Western blot analysis revealed abundant SPEC2 protein in lymphocytes, which in glutathione S-transferase-capture experiments specifically interacted with only GTP-bound Cdc42. Immunofluorescence experiments revealed that the SPEC2 protein was diffusely localized in the cytoplasm and at the cell membrane in unstimulated Jurkat T cells and Raji B cells. Recruitment of SPEC2 within Jurkat T cells to the antigen-presenting cell interface occurred following incubation with staphylococcal enterotoxin E superantigen-loaded B cells and colocalized there with F-actin and Cdc42. T cell receptor (TCR) activation studies using anti-CD3 antibody-coated polystyrene beads showed that SPEC2 was recruited to the site of bead contact, which was not observed with anti-major histocompatibility complex antibody-coated beads. Accumulation of SPEC2 following TCR engagement occurred as early as 5 min, before obvious F-actin accumulation. Biochemical studies with Jurkat T cells demonstrated that N-terminal cysteine residues in SPEC2 were palmitoylated. Overexpression studies of the related SPEC1 showed that it also was recruited to the activated TCR. Mutational analysis revealed that localization of SPEC1 to the TCR required two N-terminal cysteine residues. Furthermore, a SPEC1 Cdc42 Rac-interacting binding mutant, containing an intact N terminus but defective in Cdc42 binding, completely blocked F-actin accumulation at the activated TCR. Taken together these results suggest that SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse.

Methylation Signals Outside the Nucleus

Ezh2 is a member of the polycomb group of proteins and functions in the control of development by catalyzing methylation of lysine residues on histone proteins, thereby causing changes in gene expression. However, Ezh2 exists in the cytoplasm as well as in the nucleus, so Su et al. explored whether the enzyme might have functions independent of its known role in modification of chromatin in the nucleus. Ezh2 has been reported to associate with the guanine nucleotide exchange factor Vav1, which is an important component of T cell signaling, particularly changes in actin polymerization in response to stimulation of the T cell receptor (TCR). Studies of mouse peripheral T cells confirmed interaction of Ezh2 with Vav. Furthermore, T cells lacking Ezh-2 had defective TCR-induced actin polymerization and an impaired proliferative response. Similarly, mouse embryo fibroblasts lacking Ezh2 showed decreased actin polymerization in response to fibroblast growth factor and failed to form surface ruffles normally seen in platelet-derived growth factor (PDGF)-treated cells. These effects could be rescued by expression of a modified form of Ezh2 that was not localized to the nucleus, supporting a role for the cytoplasmic protein independent of its nuclear effects. Proliferation in response to PDGF was also reduced in cells lacking Ezh2. The presumed cytoplasmic methylation target of Ezh2 remains unknown but appears to act at a point between TCR activation and activation of the guanosine triphosphatase (GTPase) Cdc42. Vav1, though, appears not to be modified by Ezh2. Together, the findings indicate that posttranslational modification by methylation has key regulatory roles outside the nucleus, with implications for immune responses to the TCR and cancer biology, where increased expression of Ezh2 in cancer cells is associated with increased metastatic capacity. I-h. Su, M.-W. Dobenecker, E. Dickinson, M. Oser, A. Basavaraj, R. Marqueron, A. Viale, D. Reinberg, C. Wulfing, A. Tarakhovsky, Polycomb group protein Ezh2 controls actin polymerization and cell signaling. Cell 121 , 425-436. [Online Journal]

RhoH, a hematopoietic-specific Rho GTPase, regulates proliferation, survival, migration, and engraftment of hematopoietic progenitor cells

AbstractRho guanosine triphosphatases (GT-Pases) are recognized as critical mediators of signaling pathways regulating actin assembly, migration, proliferation, and survival in hematopoietic cells. Here, we have studied a recently identified hematopoietic-specific Rho GTPase, RhoH. Unlike most members of the Rho GTPase family, RhoH is GTPase deficient and does not cycle between GTP- and guanosine diphosphate (GDP)–bound forms, suggesting that regulation of RhoH expression may be critical in its activity. We found that RhoH is expressed in murine hematopoietic progenitor cells (HPCs) and fully differentiated myeloid and lymphoid lineages. In cytokine-stimulated HPCs, knockdown of RhoH expression via RNA interference stimulates proliferation, survival, and stromal cell-derived factor-1α (SDF-1α)–induced migration in vitro. Conversely, RhoH overexpression in these cells via retrovirus-mediated gene transfer is associated with impaired activation of Rac GTPases, reduced proliferation, increased apoptosis, and defective actin polymerization and chemotaxis. In vivo, HPCs with RhoH overexpression demonstrate defective hematopoietic reconstitution capability compared with control vector-transduced cells. Our results suggest that RhoH serves as a negative regulator of both growth and actin-based function of HPCs possibly via suppression of Rac-mediated signaling.

Vav3 regulates osteoclast function and bone mass

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

The Arabidopsis KLUNKER gene controls cell shape changes and encodes the AtSRA1 homolog

The analysis of a group of seven trichome mutants in Arabidopsis, which all show distorted trichomes along with severe actin defects has revealed insight into the role of the actin cytoskeleton in cell shape control. Four of the corresponding genes encode components of a protein complex, the ARP2/3 complex that stimulates the production of 'fine actin' at active growth sites. In this study, we show that another member of the distorted group, KLUNKER (KLK), encodes the AtSRA1 homolog of Arabidopsis and that klk mutants show a similar range of cell shape defects to those of arp2/3 mutants. In animals, SRA1 regulates the activity of the ARP2/3-regulating WAVE-HSPC300 complex in a Rho-dependent manner. Our findings provide evidence that a Rho/ARP2/3 regulation pathway exists in plants.

Screening the yeast deletant mutant collection for hypersensitivity and hyper-resistance to sorbate, a weak organic acid food preservative.

Certain yeasts cause large-scale spoilage of preserved food materials, partly as a result of their ability to grow in the presence of the preservatives allowed in food and beverage preservation. This study used robotic methods to screen the collection of Saccharomyces cerevisiae gene deletion mutants for both increased sensitivity and increased resistance to sorbic acid, one of the most widely-used weak organic acid preservatives. In this way it sought to identify the non-essential, non-redundant activities that influence this resistance, activities that might be the potential targets of new preservation strategies. 237 mutants were identified as incapable of growth at pH 4.5 in presence of 2 mM sorbic acid, while 34 mutants exhibit even higher sorbate resistance than the wild-type parental strain. A number of oxidative stress-sensitive mutants, also mitochondrial mutants, are sorbate-sensitive. This appears to reflect the importance of sustaining a reducing intracellular environment (high reduced glutathione levels and NADH/NAD and NADPH/NADP ratios). Sorbate resistance is also very severely compromised in mutants lacking an acidified vacuole, in vacuolar protein sorting (vps) mutants, in mutants defective in ergosterol biosynthesis (erg mutants) and with several defects in actin and microtubule organization. Sorbate resistance is, however, elevated with the loss of the Yap5 transcription factor; with single losses of two B-type cyclins (Clb3p, Clb5p); and with loss of a plasma membrane calcium channel activated by endoplasmic reticulum stress (Cch1p/Mid1p).

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p.

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Gα subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.

Actin Reorganization Is Abnormal and Cellular ATP Is Decreased in Hailey-Hailey Keratinocytes

Actin reorganization and the formation of adherens junctions are necessary for normal cell-to-cell adhesion in keratinocytes. Hailey-Hailey disease (HHD) is blistering skin disease, resulting from mutations in the Ca2+ ATPase ATP2C1, which controls Ca2+ concentrations in the cytoplasm and Golgi of human keratinocytes. Because actin reorganization is among the first responses to raised cytoplasmic Ca2+, we examined Ca2+-induced actin reorganization in normal and HHD keratinocytes. Even though HHD keratinocytes display raised baseline cytoplasmic Ca2+, we found that actin reorganization in response to Ca2+ was impaired in HHD keratinocytes. Defects in actin reorganization were linked to a marked decrease in cellular ATP in HHD keratinocytes, which persists, in vivo, in HHD epidermis. Defective actin reorganization was reproduced in normal keratinocytes in which the intracellular ATP concentration had been lowered pharmacologically. ATP concentrations in undifferentiated keratinocytes markedly declined after extracellular Ca2+ was increased, but then recovered to a new baseline that was approximately 150% of the previous baseline. In contrast, ATP concentrations in HHD keratinocytes did not change in response to increased extracellular Ca2+. This report provides new insights into how the ATP2C1-controlled ATP metabolism mediates Ca2+-induced cell-to-cell adhesion in normal keratinocytes. In addition, these findings implicate inadequate ATP stores as an additional cause in the pathogenesis of HHD and suggest novel therapeutic options.

Nat3p and Mdm20p Are Required for Function of Yeast NatB Nα-terminal Acetyltransferase and of Actin and Tropomyosin

NatB Nalpha-terminal acetyltransferase of Saccharomyces cerevisiae acts cotranslationally on proteins with Met-Glu- or Met-Asp- termini and subclasses of proteins with Met-Asn- and Met-Met- termini. NatB is composed of the interacting Nat3p and Mdm20p subunits, both of which are required for acetyltransferase activity. The phenotypes of nat3-Delta and mdm20-Delta mutants are identical or nearly the same and include the following: diminished growth at elevated temperatures and on hyperosmotic and nonfermentable media; diminished mating; defective actin cables formation; abnormal mitochondrial and vacuolar inheritance; inhibition of growth by DNA-damaging agents such as methyl methanesulfonate, bleomycin, camptothecin, and hydroxyurea; and inhibition of growth by the antimitotic drugs benomyl and thiabendazole. The similarity of these phenotypes to the phenotypes of certain act1 and tpm1 mutants suggests that such multiple defects are caused by the lack of acetylation of actin and tropomyosins. However, the lack of acetylation of other unidentified proteins conceivably could cause the same phenotypes. Furthermore, unacetylated actin and certain N-terminally altered actins have comparable defective properties in vitro, particularly actin-activated ATPase activity and sliding velocity.

The Tap42-protein phosphatase type 2A catalytic subunit complex is required for cell cycle-dependent distribution of actin in yeast.

In Saccharomyces cerevisiae, the Tor proteins mediate a wide spectrum of growth-related cellular processes in response to nutrients. The pleiotropic role of the Tor proteins is mediated, at least in part, by type 2A protein phosphatases (PP2A) and 2A-like protein phosphatases. Tor-mediated signaling activity promotes the interaction of phosphatase-interacting protein Tap42 with PP2A and 2A-like protein phosphatases. The distinct complexes formed between Tap42 and different phosphatases mediate various cellular events and modulate phosphorylation levels of many downstream factors in the Tor pathway in a Tor-dependent and rapamycin-sensitive manner. In this study, we demonstrate that the interaction between Tap42 and the catalytic subunits of PP2A (PP2Ac) is required for cell cycle-dependent distribution of actin. We show that mutations in PP2Ac and Tap42 that perturb the interaction cause random distribution of actin during the cell cycle and that overexpression of the Rho2 GTPase suppresses the actin defects associated with the mutants. Our findings suggest that the Tap42-PP2Ac complex regulates the actin cytoskeleton via a Rho GTPase-dependent mechanism. In addition, we provide evidence that PP2A activity plays a negative role in controlling the actin cytoskeleton and, possibly, in regulation of the G(2)/M transition of the cell cycle.

Regulation of cell expansion by the DISTORTED genes in Arabidopsis thaliana: actin controls the spatial organization of microtubules.

The control of the directionality of cell expansion was investigated using a class of eight genes, the so-called DISTORTED (DIS) genes, that are required for proper expansion of leaf trichomes in Arabidopsis thaliana. By tracing the separation of latex beads placed on the trichome surface, we demonstrate that trichomes grow by diffuse rather than tip growth, and that in dis mutants deviations from the normal orientation of growth can occur in all possible directions. We could not detect any differences in intracellular organization between wild-type and dis-group mutants by electron microscopy. The analysis of double mutants showed that although the expression of the dis phenotype is generally independent of branching and endoreduplication, dis mutations act synthetically in combination lesions in the ZWI gene, which encodes a kinesin motor protein. Using a MAP4:GFP marker line, we show that the organization of cortical microtubules is affected in dis-group mutants. The finding that most dis-group mutants have actin defects suggested to us that actin is involved in organizing the orientation of microtubules. By analyzing the microtubule organization in plants treated with drugs that bind to actin, we verified that actin is involved in the positioning of cortical microtubules and thereby in plant cell expansion.