Localization Of Profilin Research Articles

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation.

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASP(pS157), but no changes in cofilin1/VASP(pS239) in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASP(pS157) or cofilin1/VASP(pS239) suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220-31. ©2016 AACR.

Microtubule-dependent localization of profilin I mRNA to actin polymerization sites in serum-stimulated cells

Specific localization of messenger RNA (mRNA) appears to be a general mechanism to accumulate certain proteins to subcellular compartments for participation in local processes, thereby maintaining cell polarity under strict spatiotemporal control. Transportation of mRNA with associated protein components (RNP granules) by the actin microfilament or the microtubule systems is one important mechanism to achieve this locally distributed protein production. Here we provide evidence for a microtubule-dependent localization of mRNA encoding the actin regulatory protein profilin to sites in mouse embryonic fibroblasts, which express enhanced actin polymerization.

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.

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 MINT- 6741685, MINT- 6741696, MINT- 6741707: Arpm1 (uniprotkb: Q8BXF8) physically interacts (MI: 0218) with Profilin-3 (uniprotkb: Q9DAD6) by antibait coimmunoprecipitation (MI: 0006) MINT- 6741718: Profilin-3 (uniprotkb: Q9DAD6) and Arpm1 (uniprotkb: Q8BXF8) colocalize (MI: 0403) by fluorescence microscopy (MI: 0416) MINT- 6741756: Profilin-1 (uniprotkb: P62962) binds (MI: 0407) to beta actin (uniprotkb: P60710) by pull-down (MI: 0096) MINT- 6741650: Arpm1 (uniprotkb: Q8BXF8) physically interacts (MI: 0218) with C030004A17Rik (uniprotkb: Q8C569), Vcp p97/p47 (uniprotkb: Q8CDG3), Kif21a (uniprotkb: Q9QXL2), Zfp291 (uniprotkb: Q501L8), Vcp (uniprotkb: Q01853) and Profilin-3(uniprotkb: Q9DAD6) by antibait coimmunoprecipitation (MI: 0006) MINT- 6741746: Profilin-3 (uniprotkb: Q9DAD6) binds (MI: 0407) to beta actin (uniprotkb: P60710) by pull-down (MI: 0096) MINT- 6741735:

Olive pollen profilin (Ole e 2 allergen) co‐localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination

Pollen allergens offer a dual perspective of study: some of them are considered key proteins for pollen physiology, but they are also able to trigger allergy symptoms in susceptible humans after coming in contact with their tissues. Profilin (Ole e 2 allergen) has been characterized, to some extent, as one of the major allergens from Olea europaea L. pollen, a highly allergenic species in the Mediterranean countries. In order to obtain clues regarding the biological role of this protein, we have analyzed both its cellular localization and the organization of actin throughout pollen hydration and early pollen tube germination. The localization of the cited proteins was visualized by confocal laser scanning microscopy immunofluorescence using different antibodies. Upon pollen hydration and pollen germination, a massive presence of profilin was detected close to the site of pollen tube emergence, forming a ring-like structure around the 'effective' apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. After using a permeabilization-enhanced protocol for immunolocalization, profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends. Noticeable accumulations of actin were observed in the cytoplasm of the pollen tube; particularly, in both the apical region and the area immediately close to the aperture. Actin filaments were not observed, probably due to the need of further enhanced fixation procedures. The ultrastructural localization of profilin showed the presence of the protein in the cytoplasm of both the mature pollen grain and the pollen tube. The results shown here could be interpreted as signs of a massive dissociation of the actin-profilin complexes, mobilization of actin monomers, and therefore, an intense activity of the actin cytoskeleton. The extensive release of allergenic proteins from the pollen grain into the surrounding aqueous media, as described here for profilin, may help us to understand the mechanisms by which these allergens might come in contact with the human mucosa, therefore triggering the symptoms of allergy.

Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells.

The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.

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.

Structure of the profilin-poly-L-proline complex involved in morphogenesis and cytoskeletal regulation.

Profilin, a ubiquitous low molecular weight (13,000-15,000 M(r)) actin binding protein, regulates the formation of F-actin structures in vivo, and is localized to specific cellular regions through interaction with proline-rich sequences. Here we report the 2.2 A X-ray structure of the complex between human platelet profilin (HPP) and a decamer of L-proline (L-Pro10). The L-Pro10 peptide adopts a left-handed type II poly-L-proline helix (PPII) and binds to a highly conserved patch of aromatic amino acids on the surface of profilin. The peptide and actin binding sites reside on orthogonal surfaces, and L-Pro10 binding does not result in a conformational rearrangement of HPP. This structure suggests a mechanism for the localization of profilin and its actin-related activities to sites of actin filament assembly in vivo.

Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum.

Pollen tubes show a rapid and dramatically polarized growth in which the actin cytoskeleton appears to play a central role. In order to understand the regulation of actin we characterized its associated protein, profilin, in pollen tubes of Lilium longiflorum. By using purified polyclonal antibodies prepared against bean root profilin [Vidali et al., 1995: Plant Physiol. 108:115-123] we detected in pollen grains and tubes two profilin polypeptides with molecular masses of 14.4 and 13.4 KDa, and an identical isoelectric point of 5.05. Profilin comprises approximately 0.47% of the total grain protein, with actin being approximately 1.4%. We were unable to detect a statistically significant profilin increase after germination, while the actin increased approximately 68%. We also spatially localized the distribution of profilin using immunocytochemistry of fixed cells at both the light and electron microscope level, and by fluorescent analog cytochemistry on live cells. The results show that profilin is evenly distributed throughout the cytoplasm and does not specifically associate with any cellular structure.

The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.

The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

Regulation of Profilin Localization in Saccharomyces cerevisiae by Phosphoinositide Metabolism

Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways. The subcellular localization of profilin was examined in the yeast Saccharomyces cerevisiae. Immunoblot analysis showed that profilin was localized in both the plasma membrane and cytosolic fractions of the cell. Actin was bound to the profilin localized in the cytosol. The association of profilin with the membrane was peripheral and mediated through interaction with phospholipid. The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles. The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles was required for maximum profilin binding. Moreover, the binding of profilin to phospholipid vesicles was dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate. The subcellular localization of profilin was examined in vivo under growth conditions (i.e. inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were depleted. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels resulted in a translocation of profilin from the plasma membrane to the cytosolic fraction. Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were replenished. These results suggested that phosphoinositide metabolism played a role in the localization of profilin.

Localization of profilin- and actin-like immunoreactivity in in vitro-germinated tobacco pollen tubes by electron microscopy after special water-free fixation techniques

Double immunogold labeling of profilin and actin was performed on ultrathin sections of in vitro germinated tobacco pollen using different anti-profilin and anti-actin antibodies. Since profilin, besides its role as an actin-binding protein, is known as an allergen, water-free fixation in p-formaldehyde vapor was used. Profilin labeling occurs throughout the cytoplasm of the pollen tube. There is no profilin in the pollen tube wall. Actin reactivity is found in the cytoplasm and extracellularly in the pollen tube wall where three out of four different anti-actin antibodies give a positive signal. This labeling of the pollen tube wall may result from a wall-bound actin, an isoform of actin not yet described or from the presence of a molecule immunologically indistinguishable from actin.

Tetrahymena profilin is localized in the division furrow.

Localization of Tetrahymena profilin was examined by an immunofluorescence method. In interphase Tetrahymena cells, immunofluorescence for profilin was diffusely distributed in the cytoplasm, while in dividing cells, additional intense fluorescence was observed in the division furrow. From the result of immunofluorescence localization using cytoskeletal cell models, a significant fraction of profilin appeared to become insoluble in association with a cytoskeletal structure just beneath the division furrow during cytokinesis, although remaining profilin existed as a soluble form in the cytoplasm. Double immunofluorescence staining with anti-profilin and anti-actin antibodies revealed that the localization of profilin in the division furrow coincided with that of contractile ring microfilaments in terms of both position and timing. This is the first report describing the coexistence of profilin with actin filaments in the division furrow, implying the possible involvement of profilin in assembly and disassembly of contractile ring microfilaments in the process of cytokinesis.