ABSTRACT
Profilin is a ubiquitously expressed protein well known as a key regulator of actin polymerisation. The actin cytoskeleton is involved in almost all cellular processes including motility, endocytosis, metabolism, signal transduction and gene transcription. Hence, profilin's role in the cell goes beyond its direct and essential function in regulating actin dynamics. This review will focus on the interactions of Profilin 1 and its ligands at the plasma membrane, in the cytoplasm and the nucleus of the cells and the regulation of profilin activity within those cell compartments. We will discuss the interactions of profilin in cell signalling pathways and highlight the importance of the cell context in the multiple functions that this small essential protein has in conjunction with its role in cytoskeletal organisation and dynamics. We will review some of the mechanisms that control profilin expression and the implications of changed expression of profilin in the light of cancer biology and other pathologies.
ABSTRACT
This review covers aspects of cofilin and profilin regulations and their influence on actin polymerisation responsible for cell motility and metastasis. The regulation of their activity by phosphorylation and nitration, miRs, PI(4,5)P2 binding, pH, oxidative stress and post-translational modification is described. In this review, we have highlighted selected similarities, complementarities and differences between the two proteins and how their interplay affects actin filament dynamics.
ABSTRACT
Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories.
Subject(s)
Cell Membrane/metabolism , Intravital Microscopy/methods , Profilins/metabolism , Single Molecule Imaging/methods , Spectrometry, Fluorescence/methods , Cell Line, Tumor , Cell Membrane/drug effects , Cytochalasin D/metabolism , Diffusion/drug effects , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Intravital Microscopy/instrumentation , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Nucleic Acid Synthesis Inhibitors/pharmacology , Profilins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Single Molecule Imaging/instrumentation , Spectrometry, Fluorescence/instrumentationABSTRACT
The objective of the present study was to identify an efficient and practical enrichment method for bovine type A spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 x 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 microg mL(-1) laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 microg mL(-1) DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia.
Subject(s)
Cattle , Cell Separation/veterinary , Spermatozoa/cytology , Spermatozoa/physiology , Animals , Cell Separation/methods , Cells, Cultured , Centrifugation , Flow Cytometry , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Laminin , Magnetics , Male , Plant Lectins , Povidone , Silicon DioxideABSTRACT
Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2-3 months (Stage 1, 10 g), 4-5 months (Stage 2, 35 g), and 6-7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.
