A Fluorophore Fusion Construct of Human Profilin I with Non-Compromised Poly(L-Proline) Binding Capacity Suitable for Imaging

The actin regulator profilin 1 is functionally associated with the mammalian centrosome

“…To further investigate the centrosomal distribution of profilin and to exclude the risk that the Ab used either cross-reacted with profilin 2, whose expression is up-regulated in B16 profilin 1 knockout cells (KO27) (31) or contained autoantibodies recognizing the centrosome (32), a fluorescent variant of profilin with citrine fused intra-molecularly was expressed in B16 cells. This chimeric citrineprofilin molecule binds actin and poly(L-proline) (31); the two principal interaction properties of profilin (27,33,34). After digitonin extraction, fixation, labeling of γ-tubulin with Ab and microscopy, the distribution of citrine-profilin to centrosomes as identified by the γ-tubulin staining was obvious, and a fine reticular profilin fluorescence juxtaposed to centrosomes was often particularly prominent ( Fig 1B).…”

“…Furthermore, reciprocal immunoprecipitation experiments combined with Western blotting demonstrated that profilin is a binding partner to components of the γ-tubulin ring complex. Finally, microtubule regrowth experiments and evaluation of de novo nucleation in wild-type B16 and the profilin-depleted B16 cells KO27 (31) disclosed that profilin is a negative regulator of microtubule nucleation.…”

“…Here we applied this protocol to study the perinuclear distribution of profilin in mouse melanoma B16 cells and were able to detect profilin by immunofluorescence microscopy in centrosomes identified by γ-tubulin. We then took advantage of the fluorescent citrine-profilin (31) to independently establish that profilin distributes to centrosomes. Furthermore, reciprocal immunoprecipitation experiments combined with Western blotting demonstrated that profilin is a binding partner to components of the γ-tubulin ring complex.…”

Profilin Isoforms in Health and Disease – All the Same but Different

“…The increasingly improving CRISPR/Casbased methods will open new avenues to study profilins by loss of function, gene-tagging, and gain of function experiments in a great variety of cell types and organisms (see for review (Pickar-Oliver and Gersbach, 2019). Particularly tagging of endogenous PFN1 will be feasible by new profilin constructs internally fused to fluorescent proteins (Nejedla et al, 2017), as they circumvent the impaired ligand binding of traditional GFP-fused PFN1 variants (Geese et al, 2000;Wittenmayer et al, 2000). A better insight into the functions of profilin and its isoforms will help us to understand fundamental cell biological mechanisms in health and disease.…”

Profilin—A master coordinator of actin and microtubule organization in mammalian cells

“…In support of this conjecture, profilin has been found to be crucial for the formation of cell protrusions such as lamellipodia and filopodia typically seen at advancing cell edges (Faust et al, 2019; Skruber et al, 2020). Moreover, reduced or completely blocked profilin expression strongly suppresses cell migration (Mouneimne et al, 2012; Nejedlá et al, 2017). Hence, its juxtamembrane location, connection to signaling cues such as phosphatidylinositol lipid turnover (Lindberg et al, 2008), and actin polymerization during protrusion formation and cell migration indicate a crucial role of profilin for spatiotemporal control of cell edge processes and directional cell migration.…”