Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion

Titin visualization in real time reveals an unexpected level of mobility within and between sarcomeres

Contrary to prior models in which titin serves as a stable scaffold in sarcomeres, sarcomeric and soluble titin exchange dynamically in myofibers when calcium levels are low

L-Plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages

Podosomes are cellular structures acting as degradation ‘hot-spots’ in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages

New insights and changing paradigms in the regulation of vitamin A metabolism in development

This is the peer reviewed version of the following article: Shannon, S. R., Moise, A. R., & Trainor, P. A. (2017). New insights and changing paradigms in the regulation of vitamin A metabolism in development. Wiley Interdisciplinary Reviews. Developmental Biology, 6(3), 10.1002/wdev.264, which has been published in final form at http://doi.org/10.1002/wdev.264 This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Vitamin A and its active metabolite retinoic acid are essential for embryonic development and adult homeostasis. Surprisingly, excess or deficiency of vitamin A and retinoic acid can cause similar developmental defects. Therefore, strict feedback and other mechanisms exist to regulate the levels of retinoic acid within a narrow physiological range. The oxidation of vitamin A to retinal has recently been established as a critical nodal point in the synthesis of retinoic acid, and over the past decade, RDH10 and DHRS3 have emerged as the predominant enzymes that regulate this reversible reaction. Together they form a codependent complex that facilitates negative feedback maintenance of retinoic acid levels and thus guard against the effects of dysregulated vitamin A metabolism and retinoic acid synthesis. This review focuses on advances in our understanding of the roles of Rdh10 and Dhrs3 and their impact on development and disease

Rôle de la L-plastine dans l'assemblage du cytosquelette cortical d'actine et la progression des cellules tumorales

L'organisation du cytosquelette d'actine en réseaux et en faisceaux est contrôlée par une multitude de protéines structurales. La modification de ces protéines contribue à des pathologies telles que le cancer où des changements structuraux et fonctionnels du cytosquelette d'actine induisant la progression et la transmission de signaux cellulaires dérégulées. Ce travail de thèse se concentre sur l étude du rôle de la L-plastine dans l'assemblage du cytosquelette cortical d actine et dans la progression des cellules tumorales.La L-plastine appartient à une grande famille de protéines de pontage de l actine qui contribue à l'invasion des cellules tumorales d'une façon phosphorylation-dépendante. La L-plastine est localisée au niveau de structures d actine membranaires impliquées dans la locomotion, l'adhésion et la défense immunitaire. Ici, nous avons étudié la dynamique de la L-plastine/actine dans les cellules vivantes en combinant la méthode de rétablissement de fluorescence après photoblanchissement (FRAP) avec la modélisation mathématique. Nous avons démontré quantitativement que la phosphorylation de la L-plastine au niveau de son résidu Ser5 augmente sa capacité d interagir avec l actine influençant de ce fait sa localisation intracellulaire. D'autre part, nos résultats ont montré que la L-plastine fixe et stabilise le cytosquelette d'actine et affecte la dynamique de l actine d'une façon phosphorylation-dépendante. Conformément au rôle de la L-plastine dans le contrôle de la dynamique de l'actine, nos résultats ont prouvé que le traitement au PMA (Phorbol 12-Myristate 13-Acetate), par l intermédiaire de la PKC, favorise la localisation de la L-plastine au niveau des protrusions membranaires et augmente sa phosphorylation au niveau du résidu Ser5 dans les cellules de cancer du sein MCF-7. Il est intéressant de noter que nos résultats indiquent qu une des isozymes de PKC d et/ou e semblent être impliquées dans la phosphorylation de la L-plastine dans les cellules cancéreuses.En utilisant des approches de biochimie, nous avons étayé la contribution de la L-plastine sur la régulation dynamique de l'actine, en illustrant son association avec un complexe protéique contenant la cortactine. Enfin, nous avons démontré la colocalisation de la L-plastine avec des marqueurs des invadosomes, et nous avons identifié un nouveau rôle phosphorylation-dépendant de la L-plastine dans la résistance à la mort cellulaire provoquée par le facteur de nécrose tumorale-alpha (TNF-a), consolidant le rôle de la L-plastine dans la progression des cellules tumorales.The overall organisation of the actin cytoskeleton into networks and bundles is controlled by a plethora of actin-binding proteins. Functional alteration of these actin-binding proteins contributes to pathologies such as cancer where structural and functional modifications of the actin cytoskeleton are linked to uncontrolled cell movement and signalling. This thesis focuses on the study of the role of L-plastin, an actin crosslinking protein, in the assembly of the cortical cytoskeleton and the progression of cancer cells. L-plastin is an actin filament bundling protein which contributes to cancer cell invasion in a phosphorylation-dependent manner. L-plastin localises to actin-rich membrane structures involved in locomotion, adhesion and immune defense. In this work, we have investigated L-plastin/actin dynamics in live cells by combining confocal microscopy-based fluorescence recovery after photobleaching (FRAP) method with mathematical modelling. We show that phosphorylation of L-plastin on residue Ser5 increases its association rate and may thereby influence its intracellular localisation and promote its capacity to dock efficiently to the actin cytoskeleton. Importantly, we provide evidence for the first time that L-plastin modulates actin dynamics and turn-over in focal adhesions and stress fibers, increasing their F-actin content by decreasing the actin dissociation rate at actin filament ends, effects which are also enhanced by Ser5 phosphorylation. Consistent with the L-plastin role in the control of actin dynamics, our data provide evidence that Phorbol 12-Myristate 13-Acetate (PMA) treatment, through activation of PKC, induces the translocation of L-plastin to de novo actin polymerisation sites in ruffling membranes and protruding spikes and significantly enhances L-plastin phosphorylation on residue Ser5 in the breast cancer cell line MCF-7. Interestingly, our results point to an important role for novel PKC isozymes (d and/or e) in regulating L-plastin phosphorylation in cancer cells. Using biochemical experiments, we further substantiate the contribution of L-plastin to actin dynamics regulation by illustrating its association with a protein complex containing the Arp2/3-complex activating protein cortactin. Finally, we provide evidence for the colocalisation of L-plastin with well characterised markers of podosomes and invadopodia, and we identified a novel phosphorylation-dependent function for L-plastin in conferring resistance to the cytotoxic effect of tumour necrosis factor-alpha (TNF-a), further supporting the role of L-plastin in cancer progression.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Vitamin A and retinoid signaling: genomic and nongenomic effects

Vitamin A or retinol is arguably the most multifunctional vitamin in the human body, as it is essential from embryogenesis to adulthood. The pleiotropic effects of vitamin A are exerted mainly by one active metabolite, all-trans retinoic acid (atRA), which regulates the expression of a battery of target genes through several families of nuclear receptors (RARs, RXRs, and PPARbeta/delta), polymorphic retinoic acid (RA) response elements, and multiple coregulators. It also involves extranuclear and nontranscriptional effects, such as the activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of several actors of RA signaling. However, vitamin A itself proved recently to be active and RARs to be present in the cytosol to regulate translation and cell plasticity. These new concepts expand the scope of the biologic functions of vitamin A and RA

A mathematical model of actin filament turnover for fitting FRAP data

A novel mathematical model of the actin dynamics in living cells under steady-state conditions has been developed for fluorescence recovery after photobleaching (FRAP) experiments. As opposed to other FRAP fitting models, which use the average lifetime of actins in filaments and the actin turnover rate as fitting parameters, our model operates with unbiased actin association/dissociation rate constants and accounts for the filament length. The mathematical formalism is based on a system of stochastic differential equations. The derived equations were validated on synthetic theoretical data generated by a stochastic simulation algorithm adapted for the simulation of FRAP experiments. Consistent with experimental findings, the results of this work showed that (1) fluorescence recovery is a function of the average filament length, (2) the F-actin turnover and the FRAP are accelerated in the presence of actin nucleating proteins, (3) the FRAP curves may exhibit both a linear and non-linear behaviour depending on the parameters of actin polymerisation, and (4) our model resulted in more accurate parameter estimations of actin dynamics as compared with other FRAP fitting models. Additionally, we provide a computational tool that integrates the model and that can be used for interpretation of FRAP data on actin cytoskeleton. © 2009 European Biophysical Societies' Association