Presence Of Profilin Research Articles

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Upregulation of Profilin 2 on HDAC6 overexpression in mouse GC-1 cells and its putative role in germ cell migration in the testis.

Previous reports from this laboratory have demonstrated the involvement of histone deacetylase 6 (HDAC6) in sperm motility. As the presence of HDAC6 has also been reported in the earlier stage germ cells, studies were undertaken to explore its role during these stages of spermatogenesis. HDAC6 was overexpressed in GC-1spg cells, which represent the stage between type B spermatogonia and primary spermatocyte, and its effect on germ cell transcriptome was investigated by microarray. Among the many transcripts that were differentially regulated, Profilin 2, reported previously as a neuronal specific isoform, was observed as one of the genes highly upregulated at the transcript level, which was further confirmed by real-time PCR, and the protein confirmed by indirect immunofluorescence (IIF). Profilin 2 colocalized with HDAC6, as seen both in GC-1 cells and sperm. On the sperm, the presence of Profilin 2 was detected throughout the flagella with its colocalization with HDAC6 seen conspicuously in the mid-piece region of the flagella. Co-immunoprecipitation studies confirmed Profilin 2 interaction with HDAC6. Docking studies using Z dock suggested the interaction of 8 residues of HDAC6 with 6 residues of Profilin 2. The novel observation of Profilin 2 in spermatogonial cells, its significant upregulation on HDAC6 overexpression and its interaction with HDAC6 suggests that HDAC6 in collaboration with Profilin 2 may play a role in regulating the movement of germ cells from one stage/compartment to the next.

Pointed-end processive elongation of actin filaments by Vibrio effectors VopF and VopL.

According to the cellular actin dynamics paradigm, filaments grow at their barbed ends and depolymerize predominantly from their pointed ends to form polar structures and do productive work. We show that actin can elongate at the pointed end when assisted by Vibrio VopF/L toxins, which act as processive polymerases. In cells, processively moving VopF/L speckles are inhibited by factors blocking the pointed but not barbed ends. Multispectral single-molecule imaging confirmed that VopF molecules associate with the pointed end, actively promoting its elongation even in the presence of profilin. Consequently, VopF/L can break the actin cytoskeleton's polarity by compromising actin-based cellular processes. Therefore, actin filament design allows processive growth at both ends, which suggests unforeseen possibilities for cellular actin organization, particularly in specialized cells and compartments.

Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers.

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.

Effects of Mechanical Stimuli on Profilin- and Formin-Mediated Actin Polymerization.

Self-assembling actin filaments not only form the basis of the cytoskeleton network in cells but also are utilized as nanosized building blocks to make novel active matter in which the dynamic polymerization and depolymerization of actin filaments play a key role. Formins belong to a main family of actin nucleation factors that bind to the barbed end of actin filaments and regulate actin polymerization through an interaction with profilin. Due to actomyosin contractility and relative rotation between formin and actin filaments, formin-dependent actin polymerization is subject to force and rotation constraints. However, it remains unclear how force and rotation constraints affect formin-dependent actin polymerization in the presence of profilin. Here, we show that for rotation-unconstrained actin filaments, elongation is accelerated by both force and profilin. The combined effect leads to surprisingly fast actin elongation that can approach the diffusion-limited rate at forces of a few piconewtons. The elongation of rotation-constrained filaments is also accelerated by profilin but is insensitive to applied force. We show that FH2, the main actin binding domain, plays the primary mechanosensing role. Together, the findings not only significantly advance our understanding of the mechanochemical regulation of formin-mediated actin polymerization in cells but also can potentially be utilized to make novel actin-based active matter.

Drosophila and human FHOD family formin proteins nucleate actin filaments

Formins are a conserved group of proteins that nucleate and processively elongate actin filaments. Among them, the formin homology domain-containing protein (FHOD) family of formins contributes to contractility of striated muscle and cell motility in several contexts. However, the mechanisms by which they carry out these functions remain poorly understood. Mammalian FHOD proteins were reported not to accelerate actin assembly in vitro; instead, they were proposed to act as barbed end cappers or filament bundlers. Here, we show that purified Drosophila Fhod and human FHOD1 both accelerate actin assembly by nucleation. The nucleation activity of FHOD1 is restricted to cytoplasmic actin, whereas Drosophila Fhod potently nucleates both cytoplasmic and sarcomeric actin isoforms. Drosophila Fhod binds tightly to barbed ends, where it slows elongation in the absence of profilin and allows, but does not accelerate, elongation in the presence of profilin. Fhod antagonizes capping protein but dissociates from barbed ends relatively quickly. Finally, we determined that Fhod binds the sides of and bundles actin filaments. This work establishes that Fhod shares the capacity of other formins to nucleate and bundle actin filaments but is notably less effective at processively elongating barbed ends than most well studied formins.

WH2 and proline‐rich domains of WASP‐family proteins collaborate to accelerate actin filament elongation

WASP‐family proteins are known to promote assembly of branched actin networks by stimulating the filament‐nucleating activity of the Arp2/3 complex. Here, we show that WASP‐family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP‐family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline‐rich sequence that binds profilin–actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline‐rich sequences are required to support polymerase activity by (i) bringing polymerization‐competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin–actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP‐family proteins that create it. Collaboration between WH2 and proline‐rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP‐family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.

A Diaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis

Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.

Profilin Binding Modulates the Aggregation and Phase Separation of Huntingtin N-Terminal Fragments via Polyphasic Linkage

Neurotoxic aggregates formed by N-terminal fragments of the mutant huntingtin protein (Htt-NTFs) are associated with the onset and progression of Huntington's disease. Htt-NTFs encompass expanded polyglutamine (polyQ) tracts that are flanked on the N-terminus by a 17-residue stretch and on the C-terminus by a 38-residue proline-rich stretch (C38). Htt-NTF aggregation and toxicity in cells is diminished by direct interactions with profilin, which is an abundant actin-binding protein that also binds polyproline ligands through a distinct binding site.Htt-NTF aggregation leads to the formation of soluble spherical aggregates (30-50 nm in diameter) and insoluble fibrillar deposits. These aggregates constitute distinct phases and are characterized by saturation concentrations, i.e., threshold concentrations that must be crossed in order for the corresponding phases to form. Using a combination of in vitro measurements we have demonstrated that profilin binding lowers the driving forces for Htt-NTF aggregation and phase separation via polyphasic linkage, a phenomenon whereby distinct saturation concentrations are shifted upwards by an order of magnitude. Accordingly, in the presence of profilin, a higher total concentration of Htt-NTFs is required to cross a given phase boundary. Our binding assays reveal that profilin binds preferentially to Htt-NTF monomers and small soluble oligomers. This involves specific interactions between profilin and the polyproline modules of C38 and possibly non-specific interactions between profilin and polyQ via an avidity effect.Our data provide a biophysical explanation for the observed cellular effects of profilin on Htt-NTF aggregation. Increases in saturation concentrations through polyphasic linkage might be a general mechanism for lowering the driving forces for aggregation and phase separation of aggregation-prone proteins, and may contribute to the protein homeostasis mechanisms used by cells to cope with protein aggregation stress.

Cross-Reactivity Among Pho d 2 and Profilins from Different Pollen Allergenic Sources

RationaleProfilins are considered as an important panallergen being the IgE cross-reactivity among these proteins associated with multiple pollen sensitization. Sequence homology among pollen profilins is very high, ranging 67.4-89.5%. The objective was to evaluate the cross-reactivity among profilin from palm tree pollen (Phoenix dactylifera) and other pollen allergenic sources where profilin has been described.MethodsPalm tree profilin (Pho d 2) was purified by affinity chromatography in a poly-L-proline column, sequenced by LC/MS-MS and used to produce rabbit polyclonal antibodies. Identification of profilin in eleven pollen extracts (Ambrosia artemisiifolia, Artemisia vulgaris, Betula alba, Chenopodium album, Cynodon dactylon, Helianthus annus, Mercurialis perennis, Olea europaea, Parietaria judaica, Phleum pratense and Plantago lanceolata) was carried out by IgG ELISA and immunoblot using the polyclonal antibodies. Cross-reactivity studies were performed by IgG immunoblot inhibition assays, using the purified profilin for inhibition.ResultsHighly purified profilin (>95%) from palm tree pollen (Pho d 2) with a molecular weight of 14 kDa was obtained. Identity and purity of profilin was confirmed by LC/MS-MS. Presence of profilin was confirmed in all the extracts, except Chenopodium, by direct ELISA (O.D.>0.1). Identification of profilin by IgG immunoblot, and cross-reactivity with Pho d 2 by immunoblot-inhibition, was observed in Ambrosia, Betula, Cynodon, Helianthus, Mercurialis, Plantago and Phleum extracts.ConclusionsCross-reactivity among Pho d 2 and other profilins was demonstrated in Ambrosia, Betula, Cynodon, Helianthus, Mercurialis, Plantago and Phleum extracts by immunoblot inhibition studies. Purified Pho d 2 could be used for diagnosis and treatment of pollen profilin sensitization. RationaleProfilins are considered as an important panallergen being the IgE cross-reactivity among these proteins associated with multiple pollen sensitization. Sequence homology among pollen profilins is very high, ranging 67.4-89.5%. The objective was to evaluate the cross-reactivity among profilin from palm tree pollen (Phoenix dactylifera) and other pollen allergenic sources where profilin has been described. Profilins are considered as an important panallergen being the IgE cross-reactivity among these proteins associated with multiple pollen sensitization. Sequence homology among pollen profilins is very high, ranging 67.4-89.5%. The objective was to evaluate the cross-reactivity among profilin from palm tree pollen (Phoenix dactylifera) and other pollen allergenic sources where profilin has been described. MethodsPalm tree profilin (Pho d 2) was purified by affinity chromatography in a poly-L-proline column, sequenced by LC/MS-MS and used to produce rabbit polyclonal antibodies. Identification of profilin in eleven pollen extracts (Ambrosia artemisiifolia, Artemisia vulgaris, Betula alba, Chenopodium album, Cynodon dactylon, Helianthus annus, Mercurialis perennis, Olea europaea, Parietaria judaica, Phleum pratense and Plantago lanceolata) was carried out by IgG ELISA and immunoblot using the polyclonal antibodies. Cross-reactivity studies were performed by IgG immunoblot inhibition assays, using the purified profilin for inhibition. Palm tree profilin (Pho d 2) was purified by affinity chromatography in a poly-L-proline column, sequenced by LC/MS-MS and used to produce rabbit polyclonal antibodies. Identification of profilin in eleven pollen extracts (Ambrosia artemisiifolia, Artemisia vulgaris, Betula alba, Chenopodium album, Cynodon dactylon, Helianthus annus, Mercurialis perennis, Olea europaea, Parietaria judaica, Phleum pratense and Plantago lanceolata) was carried out by IgG ELISA and immunoblot using the polyclonal antibodies. Cross-reactivity studies were performed by IgG immunoblot inhibition assays, using the purified profilin for inhibition. ResultsHighly purified profilin (>95%) from palm tree pollen (Pho d 2) with a molecular weight of 14 kDa was obtained. Identity and purity of profilin was confirmed by LC/MS-MS. Presence of profilin was confirmed in all the extracts, except Chenopodium, by direct ELISA (O.D.>0.1). Identification of profilin by IgG immunoblot, and cross-reactivity with Pho d 2 by immunoblot-inhibition, was observed in Ambrosia, Betula, Cynodon, Helianthus, Mercurialis, Plantago and Phleum extracts. Highly purified profilin (>95%) from palm tree pollen (Pho d 2) with a molecular weight of 14 kDa was obtained. Identity and purity of profilin was confirmed by LC/MS-MS. Presence of profilin was confirmed in all the extracts, except Chenopodium, by direct ELISA (O.D.>0.1). Identification of profilin by IgG immunoblot, and cross-reactivity with Pho d 2 by immunoblot-inhibition, was observed in Ambrosia, Betula, Cynodon, Helianthus, Mercurialis, Plantago and Phleum extracts. ConclusionsCross-reactivity among Pho d 2 and other profilins was demonstrated in Ambrosia, Betula, Cynodon, Helianthus, Mercurialis, Plantago and Phleum extracts by immunoblot inhibition studies. Purified Pho d 2 could be used for diagnosis and treatment of pollen profilin sensitization. Cross-reactivity among Pho d 2 and other profilins was demonstrated in Ambrosia, Betula, Cynodon, Helianthus, Mercurialis, Plantago and Phleum extracts by immunoblot inhibition studies. Purified Pho d 2 could be used for diagnosis and treatment of pollen profilin sensitization.

Drebrin is a Leaky Capper of Actin Filaments Modulating the Effects of Formins

Drebrin A is neuronal F-actin-binding protein enriched in dendritic spines, which are playing a major role in synaptic plasticity. Molecular cross-talk between drebrin and key neuronal actin-binding proteins is believed to be a part of the cytoskeleton-modulating pathway that is implicated also in synaptic dysfunction in Alzheimer's disease (AD), Down syndrome, epilepsy and normal aging. However, drebrin effects on actin dynamics and its interplay with main neuronal ABPs (such as severing proteins, cappers, elongation factors) are yet to be clarified. Here, we show that drebrin A is a leaky capper of actin filaments. Solution assays monitoring pyrene fluorescence of actin indicate that drebrin affects barbed- but not pointed end polymerization. Real time TIRF assays revealed >2-fold inhibition of F-actin elongation rate by drebrin A. Such inhibition was observed also in the presence of profilin. Moreover, consistent with its capping activity, drebrin inhibits F-actin annealing. These results called for examination of drebrin effects on barbed end binding factors, such as formins. Pyrene fluorescence assays show inhibition of formin-induced actin polymerization by drebrin A in the presence of profilin. The mechanism of such inhibition and its biological implications will be discussed.

The effect of ADF/cofilin and profilin on the dynamics of monomeric actin

The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.

Tension modulates actin filament polymerization mediated by formin and profilin

Formins promote processive elongation of actin filaments for cytokinetic contractile rings and other cellular structures. In vivo, these structures are exposed to tension, but the effect of tension on these processes was unknown. Here we used single-molecule imaging to investigate the effects of tension on actin polymerization mediated by yeast formin Bni1p. Small forces on the filaments dramatically slowed formin-mediated polymerization in the absence of profilin, but resulted in faster polymerization in the presence of profilin. We propose that force shifts the conformational equilibrium of the end of a filament associated with formin homology 2 domains toward the closed state that precludes polymerization, but that profilin-actin associated with formin homology 1 domains reverses this effect. Thus, physical forces strongly influence actin assembly by formin Bni1p.

Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly

Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. The mechanism of actin assembly is highly conserved over evolution. APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla.

FMNL2 Drives Actin-Based Protrusion and Migration Downstream of Cdc42

SummaryCell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE [1]. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex [2]. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2 [3], accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.

Barriers to Diabetes Prevention and Control Among American Indians

North Carolina's American Indian population experiences a disproportionate diabetes burden, in terms of both a high prevalence of the disease and excess diabetes-related death and disability. Concerted efforts need to be made to provide culturally appropriate and easily accessible education, health care, and health-promoting resources in these vulnerable communities.

ATP Hydrolysis Energy Transfer in the Profilin-Mediated Actin Polymerization

Profilin regulates actin polymerization in cells playing important role in cell motility and division. Actin polymerization involves ATP hydrolysis which occurs both in the absence and the presence of profilin. There is a hypothesis suggested in literature that profilin promotes actin polymerization through direct transfer of the energy of this spontaneous hydrolysis to polymerization utilizing tight coupling of the hydrolysis and corresponding polymerization events. Recently we suggested an alternative hypothesis based on an indirect energy transfer and pointed out that recent experimental and theoretical findings require re-evaluation of the direct transfer hypothesis.Our thermodynamically rigorous model of actin steady state dynamics in the presence of profilin describes all events in terms of chemical reactions and allows both energy transfer mechanisms, each corresponding to certain ranges of the rate constants (parameters) for these reactions. In fact, the difference between the two mechanisms is defined by a single ratio r of the two rates: the rate of hydrolysis by the profilin-and-ATP-bound subunit at the filament end, and the rate of dissociation of this subunit (in complex with profilin) from the end. The direct transfer cannot exist unless r>1 while indirect transfer can occur at any r.Our model predicts specific shapes for the dependence of actin critical concentration on profilin concentration depending on the ratio r. For values r>1, curves have peaks, which persist for wide ranges of other model parameters. Our experimental dependence (obtained with our new technique) shows no peak but a steady decline, contrary to the direct transfer hypothesis. This result provides strong support for our indirect transfer hypothesis.Moreover, using our theory and routine experimental techniques we determined relative activities of the two specific molecular mechanisms of profilin action (both based on indirect transfer) predicted earlier with our model.

Differential Effects of Crowding Reagents on the Interaction of Profilin and Thymosin Beta 4 with Actin

Actin polymerization in vivo is spatially organized, with dynamic actin polymerization occurring at the leading edge of a motile cell, and occurs in the context of molecular crowding effects, including osmolyte concentration, that also vary with location. The current study tests the hypothesis that molecular crowding could influence interactions of actin with actin-regulatory proteins so as to shift the pool of actin monomers to a polymerization competent state within the active polymerization zone. In support of this hypothesis, we found that a naturally occurring osmolyte trimethylamine N-oxide (TMAO) dramatically increases affinity, KT, of the actin sequestering protein thymosin b4 (TB4) to monomeric actin and significantly decreases barbed end actin critical concentration Ac in presence of ATP. The N- and C-terminal alpha helices in TB4 helices are known to be unstructured in aqueous solution and to adopt helical conformation in organic solvents or upon binding to actin. The effects of TMAO are consistent with a decrease in free energy of the actin-TB4 interaction through facilitating alpha helix formation. The effect of TMAO on the affinity, KP, of ATP-G-actin to a globular actin regulating protein, profilin, is much weaker, as might be expected based on structural considerations. Unlike TMAO, polyethylene glycol (PEG) affects Ac, KT, and KP by similar extents. We also found that the TMAO facilitates ternary complex formation between actin, profilin, and TB4 which may very significantly increase the amount of unpolymerized actin in presence of profilin and TB4, and therefore inhibit actin polymerization. We conclude that spatial variation in osmolytes could, in principle, explain how actin sheds TB4 prior to polymerization.

Observation of Individual Actin Filaments Reveals that ATP Hydrolysis is a Random Mechanism

After their polymerization into a filament, actin monomers hydrolyze their ATP and release the resulting inorganic phosphate (Pi). This phenomenon is of primary importance for the filament's assembly dynamics, as well as for its interaction with various regulatory proteins. However, the long debated question of whether Pi release follows a random or a vectorial mechanism still awaits conclusive experimental data.We have developed a novel experimental approach, combining microfluidics, optical tweezers and fluorescence microscopy, in order to manipulate and observe individual actin filaments in a controlled biochemical environment. Our data provides a direct measurement of the Pi profile in actin filaments, and clearly shows that Pi release occurs according to a random mechanism.In addition, using the same experimental setup, we have monitored the Pi profile in filaments polymerized in presence of profilin, and we have measured the impact of profilin on the kinetics of filament disassembly. We show that profilin activity is not coupled to ATP cleavage, but that it accelerates Pi release at the barbed end of actin filaments.