Actin-binding Protein Profilin Research Articles

The actin binding protein profilin 1 localizes inside mitochondria and is critical for their function.

The monomer-binding protein profilin 1 (PFN1) plays a crucial role in actin polymerization. However, mutations in PFN1 are also linked to hereditary amyotrophic lateral sclerosis, resulting in a broad range of cellular pathologies which cannot be explained by its primary function as a cytosolic actin assembly factor. This implies that there are important, undiscovered roles for PFN1 in cellular physiology. Here we screened knockout cells for novel phenotypes associated with PFN1 loss of function and discovered that mitophagy was significantly upregulated. Indeed, despite successful autophagosome formation, fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells accumulate depolarized, dysmorphic mitochondria with altered metabolic properties. Surprisingly, we also discovered that PFN1 is present inside mitochondria and provide evidence that mitochondrial defects associated with PFN1 loss are not caused by reduced actin polymerization in the cytosol. These findings suggest a previously unrecognized role for PFN1 in maintaining mitochondrial integrity and highlight new pathogenic mechanisms that can result from PFN1 dysregulation.

Actin Filament Barbed-End Depolymerization by Combined Action of Profilin, Cofilin, and Twinfilin

Cellular actin dynamics results from the collective action of hundreds of regulatory proteins, majority of which target actin filaments at their barbed ends. Three key actin binding proteins—profilin, cofilin, and twinfilin—individually depolymerize filament barbed ends. Notwithstanding recent leaps in our understanding of their individual action, how they collectively regulate filament dynamics remains an open question. In the absence of direct and simultaneous visualization of these proteins at barbed ends, gaining mechanistic insights has been challenging. We have here investigated multicomponent dynamics of profilin, cofilin, and twinfilin using a hybrid approach that combines high-throughput single filament experiments with theory. We discovered that while twinfilin competes with profilin, it promotes binding of cofilin to filament sides. Interestingly, contrary to previous expectations, we found that profilin and cofilin can simultaneously bind the same filament barbed end, resulting in its accelerated depolymerization. Our study reveals that pairwise interactions can effectively capture depolymerization dynamics in simultaneous presence of all three proteins. We thus believe that our approach of employing a theory-experiment dialog can potentially help decipher multicomponent regulation of actin dynamics. Published by the American Physical Society 2024

Phosphorylation of the actin-binding protein profilin2a at S137 modulates bidirectional structural plasticity at dendritic spines.

Background: Synaptic plasticity requires constant adaptation of functional and structural features at individual synaptic connections. Rapid re-modulation of the synaptic actin cytoskeleton provides the scaffold orchestrating both morphological and functional modifications. A major regulator of actin polymerization not only in neurons but also in various other cell types is the actin-binding protein profilin. While profilin is known to mediate the ADP to ATP exchange at actin monomers through its direct interaction with G-actin, it additionally is able to influence actin dynamics by binding to membrane-bound phospholipids as phosphatidylinositol (4,5)-bisphosphate (PIP2) as well as several other proteins containing poly-L-proline motifs including actin modulators like Ena/VASP, WAVE/WASP or formins. Notably, these interactions are proposed to be mediated by a fine-tuned regulation of post-translational phosphorylation of profilin. However, while phosphorylation sites of the ubiquitously expressed isoform profilin1 have been described and analyzed previously, there is still only little known about the phosphorylation of the profilin2a isoform predominantly expressed in neurons. Methods: Here, utilizing a knock-down/knock-in approach, we replaced endogenously expressed profilin2a by (de)phospho-mutants of S137 known to alter actin-, PIP2 and PLP-binding properties of profilin2a and analyzed their effect on general actin dynamics as well as activity-dependent structural plasticity. Results and Discussion: Our findings suggest that a precisely timed regulation of profilin2a phosphorylation at S137 is needed to mediate actin dynamics and structural plasticity bidirectionally during long-term potentiation and long-term depression, respectively.

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu2+ from actin. Altogether, our data indicate that purified recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments.

Regulation of processive actin filament elongation mediated by formin.

The formin family of proteins directs the assembly of unbranched actin filaments that are incorporated into a diverse set of higher-order cytoskeletal structures, including cytokinetic rings, filopodia and stress fibers. Formins mediate polymerization by binding the barbed end with their dimeric formin homology 2 (FH2) domains, which processively step onto incoming actin monomers to incorporate them into the filament. By remaining bound to filaments over thousands of cycles of subunit addition, formins specify changes in filament lengths while protecting the barbed ends from capping. Formin isoforms have been shown to possess a range of processive properties, which are readily modulated by changes in ionic strength, the concentrations of actin and the actin-binding protein profilin, and the application of force. However, despite its critical importance to filament assembly, the relationship between a formin's polymerization activity and its processivity remains poorly understood. To address this question, we investigated the mechanism of processive elongation mediated by the budding yeast formin Bni1p. Using in vitro assays to reconstitute polymerization under a range of conditions, we found that processive association of Bni1p with filament ends is regulated by both the rate and the mechanism by which actin monomers are added to the barbed end. As a result, the rate at which Bni1p dissociates from barbed ends and the lengths of the filaments it polymerizes are regulated differently by the polymerization conditions. This suggests that formin-mediated elongation and processivity can be independently tuned to generate networks of actin filaments with defined lengths.

Profilin’s Affinity for Formin Regulates the Availability of Filament Ends for Actin Monomer Binding

Nucleation-promoting proteins tightly regulate actin polymerization in cells. Whereas many of these proteins bind actin monomers directly, formins use the actin-binding protein profilin to dynamically load actin monomers onto their flexible Formin Homology 1 (FH1) domains. Following binding, FH1 domains deliver profilin-actin complexes to filament ends. To investigate profilin’s role as an adaptor protein in formin-mediated elongation, we engineered a chimeric formin that binds actin monomers directly via covalent attachment of profilin to its binding site in the formin. This formin mediates slow filament elongation owing to a high probability of profilin binding at filament ends. Varying the position at which profilin is tethered to the formin alters the elongation rate by modulating profilin occupancy at the filament end. By regulating the availability of the barbed end, we propose that profilin binding establishes a secondary point of control over the rate of filament elongation mediated by formins. Profilin’s differential affinities for actin monomers, barbed ends and polyproline are thus tuned to adaptively bridge actin and formins and optimize the rate of actin polymerization.

STIP1/HOP Regulates the Actin Cytoskeleton through Interactions with Actin and Changes in Actin-Binding Proteins Cofilin and Profilin.

Cell migration plays a vital role in both health and disease. It is driven by reorganization of the actin cytoskeleton, which is regulated by actin-binding proteins cofilin and profilin. Stress-inducible phosphoprotein 1 (STIP1) is a well-described co-chaperone of the Hsp90 chaperone system, and our findings identify a potential regulatory role of STIP1 in actin dynamics. We show that STIP1 can be isolated in complex with actin and Hsp90 from HEK293T cells and directly interacts with actin in vitro via the C-terminal TPR2AB-DP2 domain of STIP1, potentially due to a region spanning two putative actin-binding motifs. We found that STIP1 could stimulate the in vitro ATPase activity of actin, suggesting a potential role in the modulation of F-actin formation. Interestingly, while STIP1 depletion in HEK293T cells had no major effect on total actin levels, it led to increased nuclear accumulation of actin, disorganization of F-actin structures, and an increase and decrease in cofilin and profilin levels, respectively. This study suggests that STIP1 regulates the cytoskeleton by interacting with actin, or via regulating the ratio of proteins known to affect actin dynamics.

Increases in mRNA and Protein Levels of the Genes for the Actin-Binding Proteins Profilin, Fascin, and Ezrin Promote Metastasis in Non-Small Cell Lung Cancer

Increases in mRNA and Protein Levels of the Genes for the Actin-Binding Proteins Profilin, Fascin, and Ezrin Promote Metastasis in Non-Small Cell Lung Cancer

Increases in mRNA and Protein Levels of the Genes for the Actin-Binding Proteins Profilin, Fascin, and Ezrin Promote Metastasis in Non-Small Cell Lung Cancer

The actin-binding proteins profilin, fascin, and ezrin were tested for involvement in metastasis of non-small cell lung cancer (NSCLC). The levels of the PFN1, FSCN1, and EZR mRNAs and respective proteins were determined by real-time PCR and Western blotting; tumor and adjacent normal lung tissue samples were obtained from 46 NSCLC patients. Patients with lymphatic metastasis had higher expression levels of the profilin, fascin, and ezrin mRNAs and the profilin and fascin proteins. Both mRNA and protein expression levels increased in patients with distant metastasis. The molecules may serve as predictors to evaluate the prognosis in NSCLC.

New slbo-Gal4 driver lines for the analysis of border cell migration during Drosophila oogenesis.

Border cell (BC) migration during Drosophila oogenesis is an excellent model for the analysis of the migratory and invasive cell behavior. Most studies on BC migration have exploited a slbo-Gal4 driver to regulate gene expression in these cells or to mark them. Here, we report that the slbo-Gal4 transgene present in the line #6458 from the Bloomington Stock Center is inserted within chickadee (chic), a gene encoding the actin-binding protein Profilin, which promotes actin polymerization and is known to be involved in cell migration. The chic6458 mutation caused by the transgene insertion behaves as a null chic allele and is homozygous lethal. To evaluate possible effects of chic6458 on the assessment of BC behavior, we generated new lines bearing the slbo-Gal4 transgene inserted into different second chromosome loci that do not appear to be involved in cell migration. Using these new lines and the slbo-Gal4-chic6458 line, we defined the functional relationships between the twinfilin (twf) and chic in BC migration. Migration of BCs is substantially reduced by mutations in twf, which encodes an actin-binding protein that inhibits actin filament assembly. The defects caused by twf mutations are significantly suppressed when the slbo-Gal4-chic6458, but not the new slbo-Gal4 drivers were used. These findings indicatetwf and chic interact and function antagonistically during BC migrationin Drosophila oogenesis.

Differential proteome analysis of pea roots at the early stages of symbiosis with nodule bacteria

In this paper, we have analyzed changes in the proteomic spectrum of pea Pisum sativum L. roots during inoculation with rhizobial bacteria with the aim of revealing new regulators of symbiosis development. To study the changes in the proteome spectrum of pea roots, a differential twodimensional (2-D) electrophoresis was performed using fluorescent labels Cy2 and Cy5. The images obtained made it possible to identify differences between the control variant (uninoculated roots) and the root variant after inoculation with Rhizobium leguminosarum bv. viciae RCAM 1026 (24 hours after treatment). 20 proteins were revealed and identified, the synthesis of which was enhanced during the inoculation of pea roots by nodule bacteria. To identify the proteins, a mass spectrometric analysis of tryptic peptides was performed on a quadrupole-time-of-flight mass spectrometer combined with a high-performance liquid chromatograph. Among such proteins, the beta-subunit of the G protein and the disulfide isomerase/phospholipase C were first found, whose function can be related to the signal regulation of symbiosis. This indicates that G-proteins and phospholipases can play a key role in the development of early stages of symbiosis in peas. Further experiments are expected to show whether the beta-subunit of the G protein interacts with the receptors to Nod factors, and how this affects the further signaling. Other proteins that might be interesting were annexin D8 and D1, protein kinase interacting with calcinerin B, actin-binding protein profilin, GTP-binding protein Ran1. They may be involved in the regulation of reactions with calcium, the reorganization of the actin cytoskeleton and other important processes in plants. The study of the role of such regulatory proteins will later become the basis for understanding the complex system of signal regulation, which is activated in pea plants by interaction with nodule bacteria.

The actin-binding protein profilin 2 is a novel regulator of iron homeostasis

AbstractCellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis-regulatory iron-responsive elements (IRE) on target messenger RNAs (mRNA). We identified profilin 2 (Pfn2) mRNA, which encodes an actin-binding protein involved in endocytosis and neurotransmitter release, as a novel IRP-interacting transcript, and studied its role in iron metabolism. A combination of electrophoretic mobility shift assay experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 3′ untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus, and midbrain) and reduction of the hepatic iron store without anemia. Despite low liver iron levels, hepatic hepcidin expression remained high, likely because of compensatory activation of hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice.

Guttiferone K suppresses cell motility and metastasis of hepatocellular carcinoma by restoring aberrantly reduced profilin 1.

Hepatocellular carcinoma (HCC) is an aggressive malignancy and the 5-year survival rate of advanced HCC is < 10%. Guttiferone K (GUTK) isolated from the Garcinia genus inhibited HCC cells migration and invasion in vitro and metastasis in vivo without apparent toxicity. Proteomic analysis revealed that actin-binding protein profilin 1 (PFN1) was markedly increased in the presence of GUTK. Over-expression of PFN1 mimicked the effect of GUTK on HCC cell motility and metastasis. The effect of GUTK on cell motility was diminished when PFN1 was over-expressed or silenced. Over-expression of PFN1 or incubation with GUTK decreased F-actin levels and the expression of proteins involved in actin nucleation, branching and polymerization. Moreover, a reduction of PFN1 protein levels was common in advanced human HCC and associated with poor survival rate. In conclusion, GUTK effectively suppresses the motility and metastasis of HCC cells mainly by restoration of aberrantly reduced PFN1 protein expression.

Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons

The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca2+-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis.

Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation.

Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits.

Amyotrophic lateral sclerosis-associated mutant profilin 1 increases dendritic arborisation and spine formation in primary hippocampal neurons

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease and familial ALS accounts for 10% of cases. The identification of familial ALS mutations in the actin-binding protein profilin 1 directly implicates actin dynamics and regulation in the pathogenesis of ALS. The mechanism by which these mutations cause ALS is unknown. In this study we show that expression of the ALS-associated actin-binding deficient mutant of PFN1 (PFN1C71G) results in increased dendritic arborisation and spine formation, and cytoplasmic inclusions in cultured mouse hippocampal neurons.

Evaluation of actin cytoskeleton in non-host resistance of pepper to Puccinia striiformis f. sp. tritici stress

Non-host resistance endows plants with immunity to most microbial pathogens. To evaluate the role of actin cytoskeleton in nonhost resistance of pepper against the wheat stripe rust, the expression levels of actin-binding protein genes Rho, profilin and actin depolymerizing factor (ADF) were detected in pepper leaves inoculated with the nonpathogen Puccinia striiformis f. sp. tritici (Pst) by qRT-PCR. In addition, actin polymerization inhibitor Latrunculin B was used to reveal how actin is involved in defense responses of the nonhost pepper against Pst infection. The results showed that Rho, profilin and actin depolymerizing factor were induced with different expression levels after inoculation with Pst. Furthermore, a reduced incidence of H2O2 accumulation and hypersensitive response occurred in pepper leaves treated with Latrunculin B compared with the control. Both gene expression and pharmacological results suggest that the actin cytoskeleton is likely to contribute to the non-host resistance of pepper against Pst, and a dynamic actin cytoskeleton is necessary for the pepper against Pst infection.

The distribution of profilin in root-tip cells of wheat seedlings exposed to enhanced UV-B radiation

ABSTRACTActin filaments play a significant role in regulating the cell cycle. The dynamic rearrangement of actin filaments are regulated by actin-binding proteins profilin (PFN). However, the distribution of PFN in root-tip cells and the role of PFN in the UV response have been unknown. Here, we observed the distribution of PFN during every stage of wheat cell mitosis in the control and UV-B treatment group, and found that PFN showed a punctuate pattern of localization around the periphery of nuclear envelopes in interphase and gathered toward the cell bipolar in prophase in the control treatment group. During metaphase, PFN presented a cage shape that is formed around the chromosomes, then appeared in the equator during anaphase, and re-distributed around the periphery of nuclear envelopes during cytokinesis. But PFN localization had changed under enhanced UV-B radiation (10.08 kJm–2 h–1), PFN displayed an annular distribution in interphase and, during metaphase, relatively large number of PFN distributed in one of the four corners or gathered in the four corners in the cell. However, two corners of PFN moved to the other two corners along the direction perpendicular to the cell-elongating axis during telophase. And these aberrant distributions of PFN were often associated with abnormal chromosome distribution.

Abstract 1444: Docosahexaenoic acid reduces cancer cell migration may link with actin binding proteins and miRNA 17-92 cluster expressions changes

Abstract Metastasis and progression, reflected by higher migration and longer cell survival, are major contributing factors in making cancers the leading cause of death worldwide. Cancer cells exhibit higher migration rates and greater cell viability facilitated by actin binding protein-mediated cytoskeletal remodeling and secondary messengers (cAMP and/or cGMP) signaling. Studies have shown that docosahexaenoic acid (DHA), a poly unsaturated fatty acid, inhibits cancer cell metastatic phenotype. Moreover, miRNA-17∼92 cluster has been reported to be highly expressed in metastasis. Therefore, we put forward a hypothesis that DHA will induce changes in actin binding protein dynamics and miRNA-17∼92 cluster expression to inhibit cancer cell migration and viability. Non-cance (MLE12) and cancer (A549) cells were treated with 8-Br-cAMP and/or DHA for 30 min, 3h, 6h and 24h. Actin binding proteins (profilin, cofilin, vimentin and gelsolin) and miRNA-17∼92 cluster expression were analyzed by western blot and qRT PCR respectively. Cell migration was estimated by wound assay and transwell apparatus. F-actin content and actin binding proteins were measured using confocal microscopy at the leading edges of wound. Proliferation and cell viability were estimated by flow cytometer, IF using ki-67, and cleaved-caspase-3 antibodies. F-actin content, cell migration and proliferation were increased while cell viability was decreased by cAMP. DHA treatment reverses the increase in actin content and cell migration in cancer cells but not in non-cancer cells. These findings correlate with changes in actin binding protein content and miRNA 17∼92 cluster expression. Thus, DHA specifically inhibits cancer cell migration, proliferation, and viability and this effect correlates with decreases in actin binding protein levels and miRNA-17∼92 cluster expression. In conclusion, DHA supplementation suppresses cellular changes related to metastatic potential in cancer cells and could provide therapeutic potential against cancer cell metastasis. Moreover, actin binding proteins and miRNA-17∼92 cluster expression could serve as innovative therapeutic targets and biomarkers for cancer progression. Citation Format: Mehboob Ali, Kathryn M. Heyob, Lynette K. Rogers. Docosahexaenoic acid reduces cancer cell migration may link with actin binding proteins and miRNA 17-92 cluster expressions changes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1444. doi:10.1158/1538-7445.AM2015-1444

Profilin isoforms modulate astrocytic morphology and the motility of astrocytic processes.

The morphology of astrocytic processes determines their close structural association with synapses referred to as the ‘tripartite synapse’. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes.Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as isoform-specific functions.