Organizational Innovation of Apical Actin Filaments Drives Rapid Pollen Tube Growth and Turning.

Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport during pollen tip growth in Arabidopsis. We found that actin filaments originating from the apical membrane form a specialized structure consisting of longitudinally aligned actin bundles at the cortex and inner cytoplasmic filaments with a distinct distribution. Using actin-based pharmacological treatments and genetic mutants in combination with FRAP (fluorescence recovery after photobleaching) technology to visualize the transport of vesicles within the growth domain of pollen tubes, we demonstrated that cortical actin filaments facilitate tip-ward vesicle transport. We also discovered that the inner apical actin filaments prevent backward movement of vesicles, thus ensuring that sufficient vesicles accumulate at the pollen tube tip to support the rapid growth of the pollen tube. The combinatorial effect of cortical and internal apical actin filaments perfectly explains the generation of the inverted "V" cone-shaped vesicle distribution pattern at the pollen tube tip. When pollen tubes turn, apical actin filaments at the facing side undergo depolymerization and repolymerization to reorient the apical actin structure toward the new growth direction. This actin restructuring precedes vesicle accumulation and changes in tube morphology. Thus, our study provides new insights into the functional relationship between actin dynamics and vesicle transport during rapid and directional pollen tube growth.

NAMPT-mediated salvage synthesis of NAD+ controls morphofunctional changes of macrophages.

Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+'s cytosolic role in the regulation of morphofunctional characteristics of macrophages.

Role for myosin-V motor proteins in the selective delivery of Kv channel isoforms to the membrane surface of cardiac myocytes.

RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Protein expression in pectoral skeletal muscle of chickens as influenced by dietary methionine.

Effects of dietary methionine (Met) on pectoralis muscle development and the effect that Met as a nutritional substrate has on protein expression of skeletal muscle cells of pectoralis muscle of chickens were evaluated in this study. Broiler chickens received a common pretest diet up to 21 d of age and were subsequently fed either a low (LM) or high Met (HM) diet (0.41 vs. 0.51% of diet) from 21 to 42 d of age. Dietary deficiency was shown in vivo judging by the depression in breast meat weight and yield when broilers were fed the LM diet. Global protein expression was analyzed by quantitative high-performance liquid chromatography nanospray ionization tandem mass spectrometry. Up- and downregulated proteins were analyzed via Ingenuity Pathways Analysis to identify the metabolic pathways affected. Four canonical pathways related to muscle development were identified as being differentially regulated between LM- and HM-fed chickens. These pathways included the citrate cycle and calcium, actin cytoskeleton, and clathrin-mediated endocytosis signaling. The HM diet may have allowed for increased muscle growth by an increased availability of nutrients to muscle cells. Although the Met supplementation was associated with enhanced breast muscle growth, contraction fiber concentrations in muscles decreased and were associated with a lower calcium transportation rate and sensitivity and with a lower energy supply. It is further suggested that increased muscle protein deposition, that was induced by Met supplementation, may have been largely due to sarcoplasmic rather myofibrillar hypertrophy.

Actin bundler PLIM2s are involved in the regulation of pollen development and tube growth in Arabidopsis.

Microspores develop inside the anther, where they are surrounded by nourishing tapetal cells. However, many cellular processes occurring during microspore development in the locule are poorly characterized. The actin cytoskeleton is known to play a crucial role in various aspects of the plant developmental process. During pollen tube tip growth, actin cytoskeleton serves as an efficient molecular transportation track, although how it functions in pollen development is unknown. The plant actin bundler PLIM2s have been shown to regulate actin bundling in different cells. Here, we investigate the biological function of three Arabidopsis pollen-specific LIM proteins, PLIM2a, PLIM2b, and PLIM2c (collectively, PLIM2s), in pollen development and tube growth. Variable degrees of suppressed expression of the PLIM2s by RNA interference resulted in aberrant phenotypes. Complete suppression of the PLIM2s totally disrupted pollen development, producing abortive pollen grains and rendering the transgenic plants sterile. Partial suppression of the PLIM2s arrested pollen tube growth to a lesser extent, resulting in short and swollen pollen tubes. Finally, the PLIM2c promoter initiated expression in pollen during stamen filament elongation, and the PLIM2c protein was located on particle structures in the developing pollen grains in Arabidopsis. These suggest that the actin bundler, PLIM2s, are an important factor for Arabidopsis pollen development and tube growth.

Proteomics displays cytoskeletal proteins and chaperones involvement in Hedyotis corymbosa-induced photokilling in skin cancer cells.

Photodynamic therapy was found to be an effective therapy for local malignant tumors. This study demonstrated that 80 µg/ml Hedyotis corymbosa extracts with 0.8 J/cm(2) fluence dose caused M21 skin cancer cell death. Photoactivated H. corymbosa-induced M21 cell death is a typical apoptosis that is accompanied by nuclear condensation, externalization of phosphatidylserine and the changes in protein expression of apoptosis-related proteins, such as Bcl-2 and caspase family members. This study applied 2D electrophoresis to analyse the proteins involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We found 12 proteins to be markedly changed. According to the results of protein sequence analysis of these altered protein spots, we identified that the expression of cytoskeletal proteins and chaperones were involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We further demonstrated that photoactivated H. corymbosa caused a significant effect on the cytoskeleton distribution and mitochondrial activity in M21 cells. Based on the above findings, this study characterized the effects and mechanisms of the photoactivated H. corymbosa-induced apoptosis in M21 skin cancer cells.

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function.

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.

What can myosin VI do in cells?

The recently solved structure of the myosin VI motor demonstrates that the unique insert at the end of the motor is responsible for the reversal of the normal myosin directionality. A second class-specific insert near the nucleotide-binding pocket contributes to myosin VI's unique kinetic tuning, allowing it to function either as an actin-based transporter or as an anchoring protein. Recent biochemical and biophysical studies have shown that the native molecule can form dimers upon clustering, and cell biological studies have demonstrated that it clearly does play both transport and anchoring roles in cells. These mechanistic insights allow us to speculate on how unusual aspects of myosin VI structure and function allow it to fill unique niches in cells.

Hypersensitivity of myofilament response to Ca2+ in association with maladaptation of estrogen-deficient heart under diabetes complication.

The amelioration of cardioprotective effect of estrogen in diabetes suggests potential interactive action of estrogen and insulin on myofilament activation. We compared Ca2+-dependent Mg2+-ATPase activity of isolated myofibrillar preparations from hearts of sham and 10-wk ovariectomized rats with or without simultaneous 8 wk-induction of diabetes and from diabetic-ovariectomized rats with estrogen and/or insulin supplementation. Similar magnitude of suppressed maximum myofibrillar ATPase activity was demonstrated in ovariectomized, diabetic, and diabetic-ovariectomized rat hearts. Such suppressed activity and the relative suppression in alpha-myosin heavy chain level in ovariectomy combined with diabetes could be completely restored by estrogen and insulin supplementation. Conversely, the myofilament Ca2+ hypersensitivity detected only in the ovariectomized but not diabetic group was also observed in diabetic-ovariectomized rats, which was restored upon estrogen supplementation. Binding kinetics of beta1-adrenergic receptors and immunoblots of beta1-adrenoceptors as well as heat shock 72 (HSP72) were analyzed to determine the association of changes in receptors and HSP72 to that of the myofilament response to Ca2+. The amount of beta1-adrenoceptors significantly increased concomitant with Ca2+ hypersensitivity of the myofilament, without differences in the receptor binding affinity among the groups. In contrast, changes in HSP72 paralleled that of maximum myofibrillar ATPase activity. These results indicate that hypersensitivity of cardiac myofilament to Ca2+ is specifically induced in ovariectomized rats even under diabetes complication and that alterations in the expression of beta1-adrenoceptors may, in part, play a mechanistic role underlying the cardioprotective effects of estrogen that act together with Ca2+ hypersensitivity of the myofilament in determining the gender difference in cardiac activation.

Use of the Ca-shortening curve to estimate the myofilament responsiveness to Ca2+ in tetanized rat ventricular myocytes.

We previously estimated the myofilament responsiveness to Ca(2+) in isolated intact ventricular myocytes, using the steady-state relationship between cytosolic Ca(2+) concentration ([Ca(2+)](i)) and cell-shortening during tetanus (Ca-L trajectory). This method was useful and easy; however, it could not be used for a high dose of Ca sensitizer because the instantaneous plots after the application of Ca sensitizer did not make a fixed point of shortening (we used 5% shortening). Therefore we must produce another method to investigate Ca(2+) responsiveness. For an estimation of a wider range of the Ca-L trajectory, we fitted the Ca-L trajectory data with the Hill equation to construct the Ca-shortening curve. To fit this curve, we measured the maximal shortening, which was on average 31.6%. The value of [Ca(2+)](i) to produce the half-maximal shortening (Ca(50)) was dose-dependently decreased by EMD57033 (sensitization). Either isoproterenol or 3-isobutyl-1-methylxanthine increased Ca(50) (desensitization) with a concomitant increase in intracellular c-AMP. EMD57439, a selective PDE-III inhibitor, did not significantly increase the c-AMP concentration and produced little change in Ca(50). These results are in agreement with previous reports with skinned or intact multicellular preparations. The Ca-shortening curve constructed in intact cardiac myocytes can be used to estimate the myofibrillar responsiveness to Ca(2+) in a wide range of [Ca(2+)](i).

Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy.

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B.

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.

Heat, phosphorus NMR and microcalorimetry in relation to the mechanism of filament sliding.

For muscle heat measurements the methods available are sensitive and rapid, and the heat is related to the chemical changes in a manner that provides a firm outline for understanding the mechanism of contraction. For example linear dependence of the shortening heat on the sarcomere length has shown that the rate of turnover of cross-bridges increases during shortening. However, heat is bound to lack specificity. In order to cope with this problem, various methods such as rigorous chemical analyses, phosphorus NMR and microcalorimetry have been introduced. As a result of ultra-rapid freezing and chemical analysis by D. R. Wilkie (Gilbert, Kretzchmar, Wilkie and Woledge, 1971), the energy balance discrepancy between (heat + work) and the amount of phosphocreatine (PCr) split emerged, i.e. the unexplained enthalpy. Calcium ions move from the sarcoplasmic reticulum to the calcium-receptive proteins in the sarcoplasm during contraction. In an attempt to find the cause of the unexplained enthalpy, microcalorimetry of calcium binding to calcium-receptive proteins has been performed. The results have shown that calcium ions dislocated between sites within the sarcoplasm on activation may produce about 1/3 of the unexplained heat. In addition calcium pump should operate by consuming PCr to relocate the calcium after the contraction. Time-resolved phosphorus NMR has also shown that a certain amount of PCr splitting continues during early minute of recovery period after the contraction without Pi released. This delayed splitting of PCr is most likely caused by the kinetic properties of the contractile proteins and can explain another 1/3 of the unexplained enthalpy. The mechanism of how muscle is regulated is another important question. Studies of calcium binding to calcium-receptive proteins in the sarcoplasm by using titration microcalorimetry has shown that troponin C has a characteristic single calcium-binding site that is most likely to be involved in the regulation of contraction.

Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments.

Cytosolic free Ca2+ and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca2+ channels, as well as channel-mediated Ca2+ influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca2+ in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca2+ channel blockers La3+ or Gd3+ and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca2+. Ca2+ fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca2+ levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca2+ increase induced by CD or CB was abolished by addition of Ca2+ channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca2+ currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca2+-permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca2+-permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca2+ channel activity and may consequently regulate cytoplasmic Ca2+.

Chronic treatment with allopurinol boosts survival and cardiac contractility in murine postischemic cardiomyopathy.

Oxidative stress is a hallmark of systemic illnesses, including heart failure. Nevertheless, the overall importance of radical production in the heart remains conjectural; is it merely a marker of illness, or can intervention alter the progression of disease? This question was addressed by blocking xanthine oxidase (XO), a superoxide-generating enzyme that is upregulated in animal models of heart failure. In a randomized prospective trial design, we administered the XO inhibitor allopurinol orally to mice that had undergone massive myocardial infarction (MI). Cardiac XO activity was elevated in untreated mice after MI; allopurinol suppressed the XO activity to levels comparable to those in sham-operated mice. Eighty-one percent of untreated mice died of advanced heart failure over 2 to 4 weeks of follow-up. Survival doubled in the allopurinol-treated mice, whereas cardiac contractile function (both in vivo and in isolated muscle) was markedly improved. Response to isoproterenol was restored to near-normal levels in the allopurinol group but was attenuated in untreated mice. Oxidative modifications to proteins were prevented in the allopurinol-treated mice. Our findings indicate that targeted blockade of just one source of oxidants, XO, impacts dramatically on the progression of postischemic cardiomyopathy in mice and prevents oxidative protein modifications.

Actin-based plasticity in dendritic spines.

The central nervous system functions primarily to convert patterns of activity in sensory receptors into patterns of muscle activity that constitute appropriate behavior. At the anatomical level this requires two complementary processes: a set of genetically encoded rules for building the basic network of connections, and a mechanism for subsequently fine tuning these connections on the basis of experience. Identifying the locus and mechanism of these structural changes has long been among neurobiology's major objectives. Evidence has accumulated implicating a particular class of contacts, excitatory synapses made onto dendritic spines, as the sites where connective plasticity occurs. New developments in light microscopy allow changes in spine morphology to be directly visualized in living neurons and suggest that a common mechanism, based on dynamic actin filaments, is involved in both the formation of dendritic spines during development and their structural plasticity at mature synapses.

Molecular motors and their role in pigmentation.

Skin pigmentation is orchestrated through a series of complementary processes. After migration of melanoblasts out of the neural crest to epidermis and hair follicle, these cells mature into melanocytes. Differentiated melanocytes produce melanin in specialized organelles, the melanosomes. Moreover, the cytoplasm of melanocytes branches into extensions, the dendrites. Via the tips of these dendrites they donate their mature melanosomes to the keratinocytes resulting in skin pigmentation. Thus, one essential part of the process of pigmentation is the translocation of melanosomes from their site of origin in the perinuclear cytoplasm towards the dendrite tips. Motor proteins are molecules which use the energy derived from ATP hydrolysis to move along cytoskeletal elements, either actin filaments or microtubules, to transport their cargo, which can be organelles, vesicles or chromosomes. This review describes the different classes of microtubule-based and actin-based motor proteins with their characteristics and functional importance in cell biology and organelle transport. Some of them will be highlighted and several recent studies in mammalian pigment cells indicating their role in pigment granule transport will be discussed. As a result of these data and previous suggestions, a model will be proposed for the possible cooperation of both systems in melanosome movement.

Disruption of endothelial microfilaments selectively reduces the transendothelial migration of monocytes.

The transendothelial migration of leukocytes (diapedesis) is a central event in inflammatory and immunological processes. Although leukocyte-endothelium interactions occurring during diapedesis have been investigated intensively, little is known about the actual transmigration and the molecular mechanisms involved. Toward this end we analyzed whether the endothelial cytoskeleton plays a direct role during the transendothelial migration of monocytes. Filter-grown monolayers of human microvascular endothelial cells (HMEC-1) were treated with cytoskeleton stabilizing or destabilizing drugs and the effect of this treatment on the transmigration of peripheral blood monocytes was analyzed in a two-chamber assay. Our results show that taxol-induced stabilization of microtubules causes a reduction of leukocyte transmigration through HMEC-1, while the opposite effect is induced by the destabilization of microtubules with colchicine or nocodazol. Disruption of microfilaments with cytochalasin B or latrunculin A, on the other hand, significantly reduces the transendothelial migration although monocyte adhesion and endothelial permeability for macromolecules are slightly increased. An active participation of the endothelial microfilament system with a direct role of unconventional, calmodulin-regulated myosins is suggested by the finding that monocyte transmigration is decreased upon treatment of the endothelial cells with the Ca2+/CaM antagonist triflouperazine.

The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle.

The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angular is Ohwi et Ohashi) epicotyls. The orientation of MTs adjacent to the outer tangential wall of the cells, which has a crossed polylamellate structure with lamellae of longitudinal cellulose microfibrils alternating with lamellae of transverse cellulose microfibrils, differed from one cell to another. Treatment with an auxin-free solution caused the accumulation of cells with longitudinal MTs and subsequent treatment with a solution that contained auxin resulted in the accumulation of cells with transverse MTs, showing that sequential treatments with auxin-free and auxin-containing solutions can synchronize the reorientation of MTs. The MTs, once reoriented from longitudinal to transverse, returned to longitudinal and then back to transverse once again, the duration of the cycle being about 6h. Gibberellic acid, known to increase the percentage of cells with transverse MTs, promoted reorientation of MTs from longitudinal to transverse and inhibited that from transverse to longitudinal. Cytochalasin D, an agent that disrupts actin filaments, speeded up the reorientation from transverse to longitudinal and slowed down that from longitudinal to transverse. It caused an increase in the percentage of cells with MTs in mixed orientation, and the percentage of such cells was highest when the percentage of cells with longitudinal MTs was decreasing and that of cells with transverse MTs was increasing.

The auxin response of actin is altered in the rice mutant Yin-Yang.

The rice mutant Yin-Yang has been selected during a screen for resistance to cytoskeletal drugs and is characterized by alterations in epidermal cell length and a precocious onset of gravitropism. The elongation response of coleoptile segments to auxin does not reveal changes of auxin sensitivity in Yin-Yang. However, in contrast to the wild type, cell elongation in Yin-Yang is highly sensitive to the actin-polymerisation blocker cytochalasin D. This increased sensitivity to cytochalasin D requires optimal concentrations of auxin to become manifest. The auxin response of actin microfilaments in epidermal cells differs between wild type and mutant. In the wild type, the longitudinal microfilament bundles become loosened in response to auxin. In the mutant, these bundles disintegrate partially and are replaced by a network of short filaments surrounding the nucleus. Several aspects of the mutant phenotype can be mimicked in the wild type by treatment with cytochalasin D. The mutant phenotype is discussed in terms of signal-dependent changes of actin dynamics and the putative role of actin during cell elongation.