Actin polymerization processes in plant cells

Growing evidence shows that the actin cytoskeleton is a key effector of signal transduction, which controls and maintains the shape of plant cells, as well as playing roles in plant morphogenesis. Recently, several signaling pathways, including those triggered by hormones, Ca(2+), and cAMP, have been reported to be connected to the reorganization of the actin cytoskeleton. The molecular mechanisms involved in such signaling cascades are, however, largely unknown. The Arabidopsis genome sequence is a valuable tool for identifying some of the highly conserved molecules that are involved in such signaling cascades. Recent work has begun to unravel these complex pathways using a panoply of techniques, including genetic analysis, live-cell imaging of intracellular actin dynamics, in vivo localization of factors that are involved in the control of actin dynamics, and the biochemical characterization of how these factors function.     

Roles of actin binding proteins in mammalian oocyte maturation and beyond

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. Another class of actin-binding proteins including cofilin, tropomyosin, myosin motors, capping proteins, tropomodulin, and Ezrin-Radixin-Moesin proteins are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. In addition, actin dynamics controlling asymmetric-symmetric transitions after fertilization is a new area of investigation. Taken together, defining the mechanisms by which actin-binding proteins regulate actin cytoskeletons is crucial for understanding the basic biology of mammalian gamete formation and pre-implantation development.     

LIM proteins: association with the actin cytoskeleton

The LIM domain is an evolutionary conserved double-zinc finger motif found in a variety of proteins exhibiting diverse biological roles. LIM domains have been observed to act as modular protein-binding interfaces mediating protein-protein interactions in the cytoplasm and the nucleus. Interaction of LIM domains with specific protein partners is now known to influence its subcellular localization and activity; however, no single binding motif has been identified as a common target for LIM domains. Several LIM domain-containing proteins associated with the actin cytoskeleton have been identified, playing a role in signal transduction and organization of the actin filaments during various cellular processes.     

Collective efforts to modulate the host actin cytoskeleton by Salmonella type III-secreted effector proteins

The hallmark of Salmonella entry into host cells is extensive rearrangements of the host actin cytoskeleton at the site of Salmonella contact with intestinal epithelial cells. SopE, SopE2 and SopB, three type III effectors of Salmonella pathogenicity island 1 (SPI-1), activate the Cdc42 and Rac1 signal transduction pathways to promote these rearrangements. SipA and SipC, two Salmonella type III-secreted actin-binding proteins, directly modulate host actin dynamics to facilitate bacterial uptake. Salmonella-induced actin cytoskeleton rearrangements are therefore the result of the coordinated action of a group of type III-secreted effector proteins.     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.     

Molecular dynamics study of interactions between polymorphic actin filaments and gelsolin segment-1

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca²⁺-regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

A mutation in the cAMP signaling pathway affects sexual development of Dictyostelium discoideum

Amoebae of cellular slime molds have two developmental modes, asexual fruiting body formation and sexual macrocyst formation. How developmental choice is made is an interesting subject of wide importance. Light exposure and dry conditions are favorable for asexual development, while conditions of darkness and high humidity are so for sexual development. In Dictyostelium discoideum , the latter conditions enhance zygote formation, which determines the fate of surrounding cells for sexual development. Here, a mutant (TMC1) defective in the post-fusion aggregation of cells during sexual development is described. This mutant is also aggregationless in asexual development, and the level of cyclic adenosine monophosphate (cAMP) receptor is reduced. Correspondingly, a series of existing mutants with defects in cAMP signaling pathways showed the same sexual phenotype as TMC1. These results suggest that molecular mechanisms of development are shared by the two alternative developmental modes.     

Fine structure of gels prepared from an actin-binding protein and actin: comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of cortical cytoplasm in amoeboid cells of Dictyostelium discoideum

We have identified the three-dimensional ultrastructure of actin gels that are formed in well-characterized cell extracts and mixtures of purified actin and the 120K actin-binding protein and compared these to the ultrastructure of the cytoplasmic matrix in regions of nonextracted Dictyostelium amoebae that are rich in actin and 120K. This ultrastructural characterization was achieved by using critical-point-dried whole-mount preparations. All three preparations--gelled extracts, purified proteins, and cortical cytoplasm--are composed of filament networks. The basic morphological feature of these networks is the presence of contacts between convergent filaments resulting in "T" or "X" shaped contacts. The finding that actin-containing gels are composed of filament networks, where the primary interaction occurs between convergent filaments, reconciles the known requirement of F actin for gelation with the amorphous appearance of these gels in thin sections. Increasing the molar ratio of 120K dimer to actin monomer increases the number of contacts between filaments per unit volume and decreases the lengths of filaments between contacts. This indicates that 120K stabilizes interactions between filaments and is consistent with biochemical evidence that 120K crosslinks actin filaments. The cortical network in situ resembles more closely networks formed in 120K-rich extracts than networks assembled in mixtures of purified 120K and actin. The heterogeneity of filament diameters and variation of network density are properties shared by extracts and the cytomatrix in situ while networks found in purified 120K-actin gels have filament diameters and densities that are more uniform. These differences are certainly due to the more complex composition of cell extracts and cortical cytoplasm as compared to that of purified 120K-actin gels.     

Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

Distribution of alkaline phosphatase in vegetative dictyostelium cells in relation to the contractile vacuole complex

The structure of the contractile vacuole complex of Dictyostelium discoideum has long been a subject of controversy. A model that originated from the work of John Heuser and colleagues described this osmoregulatory organelle as an interconnected array of tubules and cisternae the membranes of which are densely populated with vacuolar proton pumps. A conflicting model described this same organelle as bipartite, consisting of a pump-rich spongiome and a pump-free bladder, the latter membranes being identified by their alkaline phosphatase activity. In the present study we have employed an antiserum specific for Dictyostelium alkaline phosphatase to examine the distribution of this enzyme in vegetative cells. The antiserum labels puncta, probably vesicles, that lie at or near the plasma membrane and are sometimes, but only rarely, enriched near contractile vacuole membranes. We conclude that alkaline phosphatase is not a suitable marker for contractile vacuole membranes. We discuss these results in relation to the two models of contractile vacuole structure and suggest that all data are consistent with the first model.     

The nysB sunD double mutant of Dictyostelium discoideum is blocked in the acquisition of non-degradative resistance to the pea phytoalexin pisatin

Dictyostelium discoideum amoebae can be induced to acquire a non-degradative resistance to the pea phytoalexin pisatin. The nysB sunD double mutant is blocked in the acquisition of this resistance. Using parasexual genetics it has been shown that sunD is a recessive mutation linked to nysB on linkage group VI.     

Phosphoproteins in dictyostelium discoideum

The phosphoproteins of Dictyostelium discoideum were compared at different stages of development by polyacrylamide gel electrophoresis. Certain phosphoproteins of vegetative amoebae were conserved while others appeared and disappeared during development. Four major phosphoproteins with apparent subunit molecular weights of 50,000, 47,000, 38,000, and 34,000 disappeared precociously in response to exogenous cAMP. Two membranal phosphoproteins, with apparent subunit molecular weights of 80,000 and 81,000, appeared precociously in response to added cAMP. One of these phosphoproteins, molecular weight of 80,000, has been identified tentatively as the “contact site A” glycoprotein. Another membranal protein, with apparent subunit molecular weight of 42,000, unaffected in its appearance by cAMP, has been identified tentatively as phosphoactin.     

Depolarization of brain synaptosomes activates opposing factors involved in regulating levels of cytoskeletal actin

Depolarization of mouse brain synaptosomes elicits transmitter release and modifies factors that regulate cytoskeletal actin (C-actin) levels. We previously reported (Bernstein and Bamburg, J. Neurosci. 1985. 5:2565–2569) that depolarization causes a release of about 25% of the actin associated with the cytoskeleton of synaptosomal lysates. From our current studies we conclude that depolarization only transiently perturbs the balance in opposing factors which regulate C-actin levels in lysates. Prolonged incubation of the lysates permits the actin to reequilibrate so that no difference between C-actin levels of resting and depolarized synaptosomes is observed. Both the initial transient release of actin from the cytoskeleton and its reassociation with the cytoskeleton during prolonged incubation are calcium dependent and involve factors in both the cytoskeletal and soluble fractions. Depolarization initiates modifications that both increase and decrease the C-actin level probably through mechanisms involving calcium sensitive actin binding proteins.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.     

Arabidopsis capping protein senses cellular phosphatidic acid levels and transduces these into changes in actin cytoskeleton dynamics

Plants respond rapidly and precisely to a broad spectrum of developmental, biotic and abiotic cues. In many instances, signaling cascades involved in transducing this information result in changes to the cellular architecture and cytoskeletal rearrangements. Based originally on paradigms for animal cell signaling, phospholipids have received increased scrutiny as key intermediates for transmitting information to the actin cytoskeleton. Significantly, a wealth of biochemical data for plant actin-binding proteins (ABPs) demonstrates that many of these interact with phosphoinositide lipids in vitro. Moreover, phosphatidic acid (PA) has been identified not only as an abundant structural lipid in plants, but also as an intermediary in developmental and stress signaling pathways that lead to altered actin organization. Several years ago, the heterodimeric capping protein (CP) from Arabidopsis was demonstrated to bind PA and is negatively regulated by this lipid in vitro. Whether this form of regulation occurs in cells, however, remained a mystery. A new study, that combines live-cell imaging of cytoskeletal dynamics with reverse-genetic analyses in Arabidopsis, provides compelling new evidence that CP is inhibited from binding filament ends in the presence of PA in vivo. This allows rapid actin polymerization and increases in filament abundance following stimulation and could be one key factor in the physiological responses of plant cells to environmental stimuli.     

Altered morphology of vegetative amoebae induced by increased expression of the Dictyostelium discoideum ras-related gene rap1

The rap1 gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The rapl gene is expressed both during growth and development in D. discoideum. To examine the action of the Rapl protein in D. discoideum, the rap1 cDNA was expressed under the control of the inducible discoidin promoter. Treatment with conditioned media, which induces the discoidin promoter, increased Rap1 protein levels in vegetative cells approximately six fold. Overexpression of the Rapl protein correlated with the appearance of morphologically aberrant vegetative amoebae: cells were extensively spread and flattened. The distribution of F-actin was altered in these cells, with an increase in actin staining around the cell periphery. Induction of the discoidin promoter by starvation in the rapl transformants also resulted in spread flat cells. When starved D. discoideum amoebae are refed with HL5 media, the cells rapidly respond by rounding up. By contrast, the rapl transformant cells showed a pronounced delay in rounding up. Rapid tyrosine phosphorylation of a p45 protein occurred in both control cells and the rapl transformant upon refeeding, implying that the signal transduction pathway leading to tyrosine phosphorylation remained functional in the rapl transformant. We propose that the Rapl protein functions in the regulation of cell morphology in D. discoideum. © 1993Wiley-Liss, Inc.     

The actin cytoskeleton orchestra in Entamoeba histolytica

Years of evolution have kept actin conserved throughout various clades of life. It is an essential protein starring in many cellular processes. In a primitive eukaryote named Entamoeba histolytica, actin directs the process of phagocytosis. A finely tuned coordination between various actin-binding proteins (ABPs) choreographs this process and forms one of the virulence factors for this protist pathogen. The ever-expanding world of ABPs always has space to accommodate new and varied types of proteins to the earlier existing repertoire. In this article, we report the identification of 390 ABPs from Entamoeba histolytica. These proteins are part of diverse families that have been known to regulate actin dynamics. Most of the proteins are primarily uncharacterized in this organism; however, this study aims to annotate the ABPs based on their domain arrangements. A unique characteristic about some of the ABPs found is the combination of domains present in them unlike any other reported till date. Calponin domain-containing proteins formed the largest group among all types with 38 proteins, followed by 29 proteins with the infamous BAR domain in them, and 23 proteins belonging to actin-related proteins. The other protein families had a lesser number of members. Presence of exclusive domain arrangements in these proteins could guide us to yet unknown actin regulatory mechanisms prevalent in nature. This article is the first step to unraveling them.