Quantitative image mean squared displacement (iMSD) analysis of the dynamics of profilin 1 at the membrane of live cells
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.
Profilin: many facets of a small protein
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.
Global proteomic profiling of the membrane compartment of bovine testis cell populations
Spermatogonial stem cells hold enormous potential in mammalian reproductive medicine through the preservation of gametes, the restoration of fertility, enhancement of germ-lineage genetic manipulation and the improvement in our understanding of stem cell biology. Here we describe the protein profiles of the membrane compartment of bovine testicular cell isolates which were enriched for germ cells using differential plating. The isolated cells were characterised with antibodies to UCHL1 (previously known as PGP9.5) for type A spermatogonia; DDX4 (previously known as VASA) for germ cells and vimentin for Sertoli cells. Ultraconservative techniques were used to specifically isolate cell membranes, with membrane protein identifications significantly increased when compared to whole cell lysates. We utilised the filter-aided sample preparation protocol for improved digestion efficiency of membrane proteins. Using ESI-LC-MS/MS, we compared the proteins present in two cell populations. A total of 1,387 proteins were identified in bovine testis cell isolates, of which 39% were membrane associated. A total of 64 proteins were differentially expressed in the non-adhered fraction (enriched for undifferentiated germ cells) compared to the adhered fraction, of which 16 were unique to this cell population and the remaining 48 showed a two-fold change (increase when compared to the adhered cell population) as judged by spectral counting. This analysis revealed a number of candidate germ cell markers including the known markers, DDX4 and UCHL1. The proteomic profiles generated in this study support and complement transcription data on gene expression and histological levels, and reinforce the potential of proteomics in identifying and characterising the protein effectors of self-renewal and/or differentiation in stem cells.
Images used for thesis Profilin Diffusion in Live Cell Membranes
Images of GFP-profilin diffusion in MDA-MB-231 cell membranes using TIRF microscopy. Images include wild type profilin and profilin modified by specific point mutations. The cells were grown under normal culture conditions, or were treated in ways that were expected to perturb profilin diffusion patterns.
Cofilin and profilin: partners in cancer aggressiveness
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.
Claudin-8 expression in Sertoli cells and putative spermatogonial stem cells in the bovine testis
Adhesion molecules are expressed by both adult and embryonic stem cells, with different classes of adhesion molecules involved in cell-membrane and intercellular contacts. In this study the expression of the adhesion molecule claudin-8 (CLDN8), a tight-junction protein, was investigated as a potential marker for undifferentiated spermatogonia in the bovine testis. We found that CLDN8 was expressed by both spermatogonia and a subset of Sertoli cells in the bovine testis. We also showed co-expression of GFRα1 in testis cells with CLDN8 and with 'Dolichos biflorus' agglutinin-fluorescein isothiocyanate (DBA-FITC) staining. We observed co-enrichment of spermatogonia and CLDN8-expressing Sertoli cells in DBA-FITC-assisted magnetic-activated cell sorting (MACS), an observation supported by results from fluorescence-activated cell sorting analysis, which showed CLDN8-expressing cells were over-represented in the MACS-positive cell fraction, leading to the hypothesis that CLDN8 may play a role in the spermatogonial stem-cell niche.
Profilin Diffusion in Live Cell Membranes
Profilin is a ubiquitously expressed protein involved in the regulation of cell proliferation, apoptosis and motility. In vitro, it is essential in the coordination of actin filament assembly and disassembly. However, the dynamics of profilin in live cells is still largely unknown. The epithelial breast cancer cell line MDA-MB-231 was transfected with wild type GFP-profilin, or GFP-profilin mutants R88A, R88E/R136D, H119E or H133S. These mutations affect the binding of profilin to phosphoinositide lipids, phosphoinositide lipids and actin, actin, or poly-proline rich domains respectively.
Image stacks of the cell membrane were acquired by TIRF microscopy and analysed using image mean square displacement (iMSD) analysis to determine if the diffusion modes (isotropic, confined or transiently confined) and diffusion rates were altered by the perturbation of profilin interactions by the different mutations. The cells were further perturbed by treatment by the actin filament disrupting drugs Cytochalasin D and Latrunculin A, by removal of cholesterol in membrane by methyl-cyclodextrin or by serum starvation.
The diffusion of wild type profilin and the profilin mutations respond in different ways to treatments, giving information not only on profilin dynamics in the cell membrane but also on the structure and organisation of the membrane itself.