Actin Tyrosine Phosphorylation Research Articles

Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.

Microcysts: The Third Developmental Pathway of Social Amoebozoans

Microcyst formation and germination, the least well studied of the three alternative developmental pathways open to the social amoebozoans, is also the simplest since it involves single cells. While classical studies profiled the morphological and biochemical changes that characterize microcyst differentiation, recent studies have focused on the molecular and evolutionary aspects of this system. Microcyst differentiation (encystment) is primarily triggered by high osmolarity and to a lesser extent by starvation. The transformation of an amoeba into a microcyst involves the formation of a cellulose cell wall as well as specific enzymatic and biochemical changes leading to dormancy. The spherical microcyst remains dormant as long as high osmotic conditions persist. When the osmolarity decreases, germination begins during which the cell wall is digested as the microcyst swells followed by the emergence of an amoeba. This rapid event typically takes 3-6 hours. The relationship between encystment and sporulation, which has evolutionary significance, is also reviewed with particular emphasis on the role and regulation of actin tyrosine phosphorylation. In light of the recent sequencing of the genomes of microcyst-forming cellular slime mold species, which will generate more research on microcysts, this critical review covers past and recent research to set the stage for future work on this alternative developmental pathway.

Tyrosine Phosphorylation of Actin during Microcyst Formation and Germination in Polysphondylium pallidum

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. The tyrosine phosphorylation state of actin does not appear to influence the long-term viability of dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.

The Formation of Actin Rods Composed of Actin Tubules in Dictyostelium discoideum Spores

A newtype of actin rod formed in both the nucleus and the cytoplasm, as well as tyrosine phosphorylation of actin, is implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. Here the ultrastructure of the rods and their relationship to the phosphorylation of actin were examined. The rods first appeared in premature spores at the midculmination stage as bundles composed of actin tubules hexagonally cross-linked. The 13-nm-diameter bundles were composed of three actin filaments. Formation of the actin rods begins during the late culmination stage and proceeds until 2 days after completion of fruiting bodies. The physical events occur in the following order; association of several modules of bundles, close packing and decrease in diameter of actin tubules, elongation of rods across the nucleus or the cytoplasm. Actin phosphorylation levels increased at the late culmination stage and reached a maximum level 12 h later. Immediately following activation of spore germination, actin was rapidly dephosphorylated, followed shortly thereafter by the disappearance of rods. Shortened actin tubules once again became arranged in a hexagonal pattern. This hexagonal arrangement of actin tubules is possibly involved in rod formation and disappearance and does not depend upon actin phosphorylation. In contrast, rod-maturation processes may correlate with actin phosphorylation.

Regulation of Dictyostelium Protein-tyrosine Phosphatase-3 (PTP3) through Osmotic Shock and Stress Stimulation and Identification of pp130 as a PTP3 Substrate

Osmotic shock and growth-medium stimulation of Dictyostelium cells results in rapid cell rounding, a reduction in cell volume, and a rearrangement of the cytoskeleton that leads to resistance to osmotic shock. Osmotic shock induces the activation of guanylyl cyclase, a rise in cGMP mediating the phosphorylation of myosin II, and the tyrosine phosphorylation of actin and the approximately 130-kDa protein (p130). We present data suggesting that signaling pathways leading to these different responses are, at least in part, independent. We show that a variety of stresses induce the Ser/Thr phosphorylation of the protein-tyrosine phosphatase-3 (PTP3). This modification does not alter PTP3 catalytic activity but correlates with its translocation from the cytosol to subcellular structures that co-localize to endosomal vesicles. This translocation is independent of PTP3 activity. Mutation of the catalytically essential Cys to a Ser results in inactive PTP3 that forms a stable complex with tyrosine-phosphorylated p130 (pp130) in vivo and in vitro, suggesting that PTP3 has a substrate specificity for pp130. The data suggest that stresses activate several interacting signaling pathways controlled by Ser/Thr and Tyr phosphorylation, which, along with the activation of guanylyl cyclase, mediate the ability of this organism to respond to adverse changes in the external environment.

High levels of actin tyrosine phosphorylation: Correlation with the dormant state of Dictyostelium spores

ABSTRACT Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days. Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.

High levels of actin tyrosine phosphorylation: correlation with the dormant state of Dictyostelium spores.

Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days. Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.

Endogenous Autoinhibitors Regulate Changes in Actin Tyrosine Phosphorylation During Dictyostelium Spore Germination

Phosphorylation of proteins on tyrosine residues has been shown to govern many cellular processes, but little work has focused on the role of tyrosine phosphorylation during germination. Under optimal conditions, D. discoideum spores synchronously germinate each liberating a single amoeba. The total amount of phosphotyrosine containing proteins observed in spores was greatest during quiescence with a gradual decline during spore activation and emergence of nascent amoebae. During dormancy, tyrosine residues of actin were heavily phsophorylated, but they gradually underwent dephosphorylation upon spore activation and this process continued through emergence. Interestingly, an endogenous autoinhibitor(s), which blocks germination, induces tyrosine phosphorylation of actin. Conversely, the removal of the autoinhibitor(s) was followed by a decrease in phosphorylation. Thus, during germination of Dictyostelium spores, actin is dephosphorylated, with the level of phosphorylation regulated by the autoinhibitor(s) and/or the autoactivator. This change in actin phosphorylation appears to play a direct role since actin dephosphorylation and reorganization is a necessary prelude to germination.

Stress-induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to tyrosine-53 adjacent to the DNase I binding loop

Actin is known to be phosphorylated at tyrosine, serine, or threonine residues in various cells. In cells of Dictyostelium discoideum, a rise in the tyrosine phosphorylation of actin is observed in response to ATP depletion. An actin fraction rich in phosphotyrosine was obtained by chromatography on the weak anion exchanger Mono-P. Mass spectrometry and amino acid sequencing of protease cleavage products indicated that a single tyrosine residue was phosphorylated. Localization of this residue to position 53 of the actin sequence attributed the modification to a site that is critical for the capability of actin to polymerize. Induction of the tyrosine phosphorylation by heat shock and Cd 2+ ions indicates that this modification of actin is implicated in the response of Dictyostelium cells to stress.

Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells.

When oxidative phosphorylation is inhibited in cells of Dictyostelium discoideum, the phosphorylation of tyrosine residues on actin is strongly increased. This increase is fully reversible. Under the same conditions the amoeboid cells undergo a series of shape changes. Within three minutes the pseudopods are withdrawn and replaced by cell surface blebs. Subsequently, the cells are rounding up to become immobile. In parallel with the changes in cell shape, the distribution of actin filaments is grossly altered within the cells. The cortical network of actin filaments of normal cells is broken down, and the F-actin forms large, irregular clusters deep within the cytoplasm. In these clusters the actin is associated with myosin II and with the heterodimeric F-actin capping protein cap32/34. After restoration of oxidative phosphorylation the actin returns within less than four minutes to its normal cortical position. A causal relationship between tyrosine phosphorylation and changes in the distribution of actin remains to be established. The rearrangements in the actin system that result from the inhibition of oxidative phosphorylation indicate that the organisation of this system and its maintenance in a functional state depend on the continuous supply of energy by ATP.

Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes.

When Dictyostelium cells that have initiated their developmental program upon starvation are returned to growth medium, there is a rapid and transient de novo tyrosine phosphorylation of a 43-kilodalton protein. This protein was found to be actin. Most of the phosphorylation occurred in a single, minor acidic isoform of actin. Developing cells that had been returned to growth medium lost their pseudopod extensions, became round, and had reduced adhesion to the substratum. These effects occurred with kinetics that matched the increase in tyrosine phosphorylation of actin. In mutant cell lines in which the gene for the phosphotyrosine phosphatase PTP1 had been disrupted, tyrosine phosphorylation of actin was rapid and more prolonged. These cells responded with proportionally accelerated kinetics of cell rounding. Cell lines overexpressing PTP1 had diminished amplitude and duration of actin tyrosine phosphorylation and exhibited diminished cell-shape change and an accelerated return to the extended cell-shape morphology seen in starved cells.