Anja Lambrechts

The mammalian profilin isoforms display complementary affinities for PIP2 and proline‐rich sequences

We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5‐bisphosphate (PIP2) and proline‐rich peptides derived from vasodilator‐stimulated phosphoprotein (VASP) and cyclase‐associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline‐rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline‐rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly‐L‐proline, since this isoform, but not profilin II, can be eluted from a poly‐L‐proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline‐rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

Stable isotopic labeling in proteomics.

Labeling of proteins and peptides with stable heavy isotopes (deuterium, carbon‐13, nitrogen‐15, and oxygen‐18) is widely used in quantitative proteomics. These are either incorporated metabolically in cells and small organisms, or postmetabolically in proteins and peptides by chemical or enzymatic reactions. Only upon measurement with mass spectrometers holding sufficient resolution, light, and heavy labeled peptide ions or reporter peptide fragment ions segregate and their intensity values are subsequently used for quantification. Targeted use of these labels or mass tags further leads to specific monitoring of diverse aspects of dynamic proteomes. In this review article, commonly used isotope labeling strategies are described, both for quantitative differential protein profiling and for targeted analysis of protein modifications.

Listeria comet tails: the actin-based motility machinery at work

Listeria monocytogenes is a master of mimicry that uses the host cell actin system both to move within the cytoplasm of infected cells and for cell-to-cell spread. Recent studies of Listeria and similarly acting pathogens have generated leaps in our understanding of the actin-based force producing machinery. This machinery is essential for most motile properties of cells, not least for cell migration. In a minimal configuration, it consists of the Arp2/3-complex, Ena-VASP proteins, cofilin, capping protein and a nucleation-promoting factor. In this review, we discuss current models of pseudopodial protrusions and describe how the road to more complex models lies open and is already paved by recent studies using Listeria-based biomimetic motility assays.

Silencing profilin-1 inhibits endothelial cell proliferation, migration and cord morphogenesis

Expression of several actin-binding proteins including profilin-1 is up-regulated during capillary morphogenesis of endothelial cells, the biological significance of which remains unknown. Specifically, we hypothesized that profilin-1 is important for endothelial migration and proliferation. In this study, we suppressed profilin-1 expression in human umbilical vein endothelial cells by RNA-interference. Gene silencing of profilin-1 led to significant reduction in the formation of actin filaments and focal adhesions. Loss of profilin-1 expression was also associated with reduced dynamics of cell-cell adhesion. Data from both wound-healing experiments and time-lapse imaging of individual cells showed inhibition of cell migration when profilin-1 expression was suppressed. Cells lacking profilin-1 exhibited defects in membrane protrusion, both in terms of its magnitude and directional persistence. Furthermore, loss of profilin-1 expression inhibited cell growth without compromising cell survival, at least in the short-term, thus suggesting that profilin-1 also plays an important role in endothelial proliferation as hypothesized. Finally, silencing profilin-1 expression suppressed matrigel-induced early cord morphogenesis of endothelial cells. Taken together, our data suggest that profilin-1 may play important role in biological events that involve endothelial proliferation, migration and morphogenesis.

Profilin II Is Alternatively Spliced, Resulting in Profilin Isoforms That Are Differentially Expressed and Have Distinct Biochemical Properties

ABSTRACT We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.

Actin isoform expression patterns during mammalian development and in pathology: Insights from mouse models

The dynamic actin cytoskeleton, consisting of six actin isoforms in mammals and a variety of actin binding proteins is essential for all developmental processes and for the viability of the adult organism. Actin isoform specific functions have been proposed for muscle contraction, cell migration, endo- and exocytosis and maintaining cell shape. However, these specific functions for each of the actin isoforms during development are not well understood. Based on transgenic mouse models, we will discuss the expression patterns of the six conventional actin isoforms in mammals during development and adult life. Ablation of actin genes usually leads to lethality and affects expression of other actin isoforms at the cell or tissue level. A good knowledge of their expression and functions will contribute to fully understand severe phenotypes or diseases caused by mutations in actin isoforms.

Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site

BackgroundProfilin is a small cytoskeletal protein which interacts with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate (PI(4,5)-P2). Crystallography, NMR and mutagenesis of vertebrate profilins have revealed the amino acid residues that are responsible for the interactions with actin and poly(L-proline) peptides. Although Arg88 of human profilin I was shown to be involved in PI(4,5)-P2-binding, it was suggested that carboxy terminal basic residues may be involved as well.ResultsUsing site directed mutagenesis we have refined the PI(4,5)-P2 binding site of human profilin I. For each mutant we assessed the stability and studied the interactions with actin, a proline-rich peptide and PI(4,5)-P2 micelles. We identified at least two PI(4,5)-P2-binding regions in human profilin I. As expected, one region comprises Arg88 and overlaps with the actin binding site. The second region involves Arg136 in the carboxy terminal helix and neighbours the poly(L-proline) binding site. In addition, we show that adding a small protein tag to the carboxy terminus of profilin strongly reduces binding to poly(L-proline), suggesting local conformational changes of the carboxy terminal α-helix may have dramatic effects on ligand binding.ConclusionsThe involvement of the two terminal α-helices of profilin in ligand binding imposes important structural constraints upon the functions of this region. Our data suggest a model in which the competitive interactions between PI(4,5)-P2 and actin and PI(4,5)-P2 and poly(L-proline) regulate profilin functions.

Dimerization of profilin II upon binding the (GP5)3 peptide from VASP overcomes the inhibition of actin nucleation by profilin II and thymosin β4

Profilin II dimers bind the (GP5)3 peptide derived from VASP with an affinity of approximately 0.5 μM. The resulting profilin II‐peptide complex overcomes the combined capacity of thymosin β4 and profilin II to inhibit actin nucleation and restores the extent of filament formation. We do not observe such an effect when barbed filament ends are capped. Neither can profilin I, in the presence of the peptide, promote actin polymerization during its early phase consistent with a lower affinity. Since a Pro17 peptide‐profilin II complex only partially restores actin polymerization, the glycine residues in the VASP peptide appear important.

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.

Profilin-I-ligand interactions influence various aspects of neuronal differentiation

Differentiating neurons extend membrane protrusions that develop into growing neurites. The driving force for neurite outgrowth is the dynamic actin cytoskeleton, which is regulated by actin-binding proteins. In this study, we describe for the first time, the role of profilin I and its ligand interactions in neuritogenesis of PC12 cells. High-level overexpression of wild-type profilin I had an inhibitory effect on neurite outgrowth. Low levels of profilin I did not disturb this process, but these cells developed many more filopodia along the neurite shafts. Low-level overexpression of mutant forms of profilin I changed one or more aspects of PC12 differentiation. Expression of a profilin I mutant that is defective in actin binding (profilin IR74E) decreased neurite length and strongly inhibited filopodia formation. Cells expressing mutants defective in binding proline-rich ligands (profilin IW3A and profilin IR136D) differentiated faster, developed more and longer neurites and more branches. The profilin IR136D mutant, which is also defective in phosphatidylinositol 4,5-bisphosphate binding, enhanced neurite outgrowth even in the absence of NGF. Parental PC12 cells treated with the ROCK inhibitor Y27632, differentiate faster and display longer neurites and more branches. Similar effects were seen in cells expressing profilin IWT, profilin IW3A and profilin IR74E. By contrast, the profilin IR136D-expressing cells were insensitive to the ROCK inhibitor, suggesting that regulation of profilin I by phosphatidylinositol 4,5-bisphosphate metabolism is crucial for proper neurite outgrowth. Taken together, our data show the importance of the interaction of profilin I with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate in neuronal differentiation of PC12 cells.