Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression

The closely related proteins AtFH4 and AtFH8 represent the group Ie clade of Arabidopsis formin homologues. The subcellular localization of these proteins and their ability to affect the actin cytoskeleton were examined. AtFH4 protein activity was identified using fluorimetric techniques. Interactions between Arabidopsis profilin isoforms and AtFH4 were assayed in vitro and in vivo using pull-down assays and yeast-2-hybrid. The subcellular localization of group Ie formins was observed with indirect immunofluorescence (AtFH4) and an ethanol-inducible green fluorescent protein (GFP) fusion construct (AtFH8). AtFH4 protein affected actin dynamics in vitro, and yeast-2-hybrid assays suggested isoform-specific interactions with the actin-binding protein profilin in vivo. Indirect immunofluorescence showed that AtFH4 localized specifically to the cell membrane at borders between adjoining cells. Expression of an AtFH8 fusion protein resulted in GFP localization to cell membrane zones, similar to AtFH4. Furthermore, aberrant expression of AtFH8 resulted in the inhibition of root hair elongation. Taken together, these data suggest that the group Ie formins act with profilin to regulate actin polymerization at specific sites associated with the cell membrane.     

Nuclear localization of profilin III-ArpM1 complex in mouse spermiogenesis

Actin-related proteins (Arps) have been reported to be localized in the cell nucleus, and implicated in the regulation of chromatin and nuclear structure, as well as being involved in cytoplasmic functions. We demonstrate here that mouse ArpM1, which closely resembles the conventional actin, is expressed exclusively in the testis, particularly in haploid germ cells. ArpM1 protein first appears in the round spermatid and changes its localization dynamically in the nucleus during spermiogenesis. By co-immunoprecipitation analysis, profilin III was identified as ArpM1-interacting protein. Our findings suggest that the testis-specific profilin III-ArpM1 complex may be involved in conformational changes in the organization of the sperm-specific nucleus. STRUCTURED SUMMARY:     

Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells.     

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

Summary Microinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band - Rh rhodamine - SMC subsidiary mother cell     

Reversible, activity-dependent targeting of profilin to neuronal nuclei

The actin cytoskeleton in pyramidal neurons plays a major role in activity-dependent processes underlying neuronal plasticity. The small actin-binding protein profilin shows NMDA receptor-dependent accumulation in dendritic spines, which is correlated with suppression of actin dynamics and long-term stabilization of synaptic morphology. Here we show that following NMDA receptor activation profilin also accumulates in the nucleus of hippocampal neurons via a process involving rearrangement of the actin cytoskeleton. This simultaneous targeting to dendritic spines and the cell nucleus suggests a novel mechanism of neuronal plasticity in which profilin both tags activated synapses and influences nuclear events.     

Characterization of Ricinus communis phloem profilin,RcPRO1

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.     

AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament-crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin-binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament-crosslinking protein. AtFim1 binds pollen actin filaments (F-actin) in a calcium-independent manner, with an average dissociation constant (Kd) of 0.55+/-0.21 microM and with a stoichiometry at saturation of 1:4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F-actin by a calcium-independent mechanism, in contrast to crosslinking of plant actin by human T-plastin, a known calcium-sensitive actin-crosslinking protein. When micro-injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2-4 min. Using the 'nuclear displacement assay' as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)-induced depolymerization, in a dose-dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin-mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium-independent, actin filament-crosslinking protein that interacts with the actin cytoskeleton in living plant cells.     

The profilin:actin complex localizes to sites of dynamic actin polymerization at the leading edge of migrating cells and pathogen-induced actin tails

A unique set of affinity-purified anti-profilin and anti-actin antibodies generated against a covalently coupled version of the profilin:actin complex was used to assess the distribution of profilin and non-filamentous actin in mouse melanoma cells. In agreement with the profilin:actin complex being the principal source of actin for filament formation, we observed extensive co-distribution of both antibody preparations with vasodilator-stimulated phosphoprotein (VASP) and the p34 subunit of the Arp2/3 complex, both of which are components of actin polymer-forming protein complexes in the cell. This suggests that the localization of profilin and actin revealed with these antibodies in fact reflects the distribution of the profilin:actin complex rather than the two proteins separately. Significantly, protruding lamellipodia and filopodia showed intensive labeling. The two antibody preparations were also used to stain HeLa cells infected with Listeria monocytogenes or vaccinia virus. In both cases, the pattern of antibody staining of the pathogen-induced microfilament arrangement differed, suggesting a varying accessibility for the antibody-binding epitopes.     

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.     

Nuclear spectrin-like proteins are structural actin-binding proteins in plants

Background information. Although actin is a relevant component of the plant nucleus, only three nuclear ABPs (actin‐binding proteins) have been identified in plants to date: cofilin, profilin and nuclear myosin I. Although plants lack orthologues of the main structural nuclear ABPs in animals, such as lamins, lamin‐associated proteins and nesprins, their genome does contain sequences with spectrin repeats and N‐terminal calponin homology domains for actin binding that might be distant relatives of spectrin. We investigated here whether spectrin‐like proteins could act as structural nuclear ABPs in plants. Results. We have investigated the presence of spectrins in Allium cepa meristematic nuclei by Western blotting, confocal and electron microscopy, using antibodies against α‐ and β‐spectrin chains that cross‐react in plant nuclei. Their role as nuclear ABPs was analysed by co‐immunoprecipitation and IF (immunofluorescence) co‐localization and their association with the nuclear matrix was investigated by sequential extraction of nuclei with non‐ionic detergent, and in low‐ and high‐salt buffers after nuclease digestion. Our results demonstrate the existence of several spectrin‐like proteins in the nucleus of onion cells that have different intranuclear distributions in asynchronous meristematic populations and associate with the nuclear matrix. These nuclear proteins co‐immunoprecipitate and co‐localize with actin. Conclusions. These results reveal that the plant nucleus contains spectrin‐like proteins that are structural nuclear components and function as ABPs. Their intranuclear distribution suggests that plant nuclear spectrin‐like proteins could be involved in multiple nuclear functions.     

Myosin16b: The COOH-tail region directs localization to the nucleus and overexpression delays S-phase progression

Rat Myo16a and Myo16b comprise the founding members of class XVI myosin and are characterized by an N-terminal ankyrin repeat domain thought to mediate an association with protein phosphatase 1 catalytic subunits 1alpha and 1gamma. Myo16b is the principal isoform and reveals predominant expression in developing neural tissue. Here, we use COS-7 cells as a model system to develop an understanding of Myo16b function. We find that Myo16b displays predominant localization in the nucleus of cells transitioning through interphase, but is not associated with processes of mitosis. Using a panel of EGFP-Myo16b-expression plasmids in transient transfection studies, we identified the COOH-terminal residues 1616-1912 as necessary and solely sufficient to target Myo16b to the nucleus. We show that the Myo16b-tail region directs localization to a nuclear compartment containing profilin and polymerized actin, which appears to form a three-dimensional meshwork through the depth of the nucleus. Further, we demonstrate that this compartment localizes within euchromatic regions of the genome and contains proliferating cell nuclear antigen (PCNA) and cyclin A, both markers of S-phase of the cell cycle. Cells transiently expressing Myo16b or Myo16b-tail region show limited incorporation of BrdU, delayed progression through S-phase of the cell cycle, and curtailed cellular proliferation.     

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells

Summary. Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.     

The differential formation of the LINC-mediated perinuclear actin cap in pluripotent and somatic cells

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape.     

Mastoparan alters subcellular distribution of profilin and remodels F-actin cytoskeleton in cells of maize root apices

Indirect immunofluorescence localization of profilin in cells of maize root apices revealed that this abundant protein was present both in the cytoplasm and within nuclei. Nucleo-cytoplasmic partitioning of profilin exhibits tissue-specific and developmental features. Mastoparan-mediated activation of heterotrimeric G-proteins, presumably through triggering a phosphoinositide-signaling pathway based on phosphatidylinositol-4,5-bisphosphate (PIP(2)), induced relocalization of profilin from nuclei into the cytoplasm of root apex cells. In contrast, PIP(2) accumulated within nuclei of mastoparan-treated root cells. Intriguingly, cytoplasmic accumulation of profilin was associated with remodeling of F-actin arrays in root apex cells. Specifically, dense F-actin networks were dismantled and distinct actin patches became associated with the periphery of small vacuoles. On the other hand, disruption of F-actin with the G-actin sequestering agent latrunculin B does not affect the subcellular distribution of profilin or PIP(2). These data suggest that nuclear profilin can mediate a stimulus-response action on the actin cytoskeleton which is somehow linked to a phosphoinositide-signaling cascade.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

Organization of perinuclear actin in live tobacco cells observed by PALM with optical sectioning

Actin performs a wide variety of different tasks. This functional diversity may be accomplished either by the formation of different isotypes or by suitable protein decoration that regulates structure and dynamics of actin filaments. To probe for such a potential differential decoration, the actin-binding peptide Lifeact was fused to different photoactivatable fluorescent proteins. These fusions were stably expressed in Nicotiana tabacum L. cv. Bright Yellow 2 cells to follow dynamic reorganization of the actin cytoskeleton during the cell cycle. The Lifeact–monomeric variant of IrisFP fusion protein was observed to indiscriminately label both, central and cortical, actin filaments, whereas the tetrameric Lifeact–photoswitchable red fluorescent protein fusion construct selectively labeled only a specific perinuclear sub-population of actin. By using photoactivated localization microscopy, we acquired super-resolution images with optical sectioning to obtain a 3D model of perinuclear actin. This novel approach revealed that the perinuclear actin basket wraps around the nuclear envelope in a lamellar fashion and repartitions toward the leading edge of the migrating nucleus. Based on these data, we suggest that actin that forms the perinuclear basket differs from other actin assemblies by a reduced decoration with actin binding proteins, which is consistent with the differential decoration model.     

Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth

In growing Arabidopsis root hairs, the nucleus locates at a fixed distance from the apex, migrates to a random position during growth arrest, and moves from branch to branch in a mutant with branched hairs. Consistently, an artificial increase of the distance between the nucleus and the apex, achieved by entrapment of the nucleus in a laser beam, stops cell growth. Drug studies show that microtubules are not involved in the positioning of the nucleus but that subapical fine F-actin between the nucleus and the hair apex is required to maintain the nuclear position with respect to the growing apex. Injection of an antibody against plant villin, an actin filament-bundling protein, leads to actin filament unbundling and movement of the nucleus closer to the apex. Thus, the bundled actin at the tip side of the nucleus prevents the nucleus from approaching the apex. In addition, we show that the basipetal movement of the nucleus at root hair growth arrest requires protein synthesis and a functional actin cytoskeleton in the root hair tube.     

Live imaging reveals that the Drosophila actin-binding ERM protein, moesin, co-localizes with the mitotic spindle

Members of the vertebrate ezrin-radixin-moesin (ERM) protein family crosslink the actin cytoskeleton and the cell membrane and are, therefore, considered cytoplasmic regulators of cell adhesion, cell movement and membrane trafficking. Here we demonstrate that besides its cytoplasmic functions Drosophila moesin, the only ERM protein in Drosophila melanogaster, exhibits a dynamic cell cycle-dependent nuclear localization. In a small fraction of cells and at a low level, moesin can be detected in interphase nuclei in regions complementary to the chromatin; its level rapidly increases during prophase and it co-localizes with the actin network surrounding the mitotic spindles throughout mitosis. We also found that the predicted single nuclear localization signal in moesin is not necessary for the nuclear accumulation of the protein. FRAP experiments confirmed this finding and further revealed that the mitotic localization of moesin is highly dynamic. Immuno-histochemical staining for moesin demonstrated the existence of spindle association in wild-type embryos. The biological relevance of this phenomenon is indicated by the mitotic phenotypes detected in S2 cells treated with moesin RNAi, and awaits future exploration.     

In vivo importance of actin nucleotide exchange catalyzed by profilin

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.     

Microinjection of profilins from different sources into the green algaMicrasterias causes transient inhibition of cell growth

Summary Recombinant profilins from different sources (Betula verrucosa, Schizosaccharomyces pombe, Acanthamoeba castellani, or man) cause marked effects on cell growth and morphogenesis when microinjected into growing cells of the green algaMicrasterias denticulata. Whereas control injections with -lactoglobulin only result in a slight delay of cell growth, when profilin is injected cell differentiation ceases and only resumes about 1 to 2 h after the injection, depending on the dose. The resulting cell does not show any malformations, but is reduced in size and retarded in differentiation compared to controls. As a consequence of the profilin microinjection the pattern of cytoplasmic streaming and cytoplasmic structure are also altered. Gelsolin, injected for comparison, leads to minor retardation of cell development but produces less marked effects than profilin. Microinjection of fluorescently labeled profilin shows even distribution throughout the cytoplasm and more intense fluorescence in the nucleus. Electron microscopical investigations of cells fixed immediately after profilin injection show a normal distribution of dictyosomes, ER cisternae, microtubules, and secretory vesicles compared to noninjected controls at the same developmental stage. Our results indicate that disturbance of the natural actin turnover by the injection of actin-binding proteins strongly affects development ofMicrasterias, corroborating a key role of actin in the morphogenetic process.