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Molecular mechanisms in the organization of specialised domains of the sarcoplasmic reticulum of muscle cells.
One of the major questions of current research is to define the subcellular localization of many proteins in cells and how they eventually interact to generate complex functional structure. The sarcoplasmic reticulum of muscle cells is a clear example of how vertebrate cells are able to organize distinct subcellular regions where proteins selectively segregate to form specialized functional domains.
a) mechanisms for delivering proteins to sub-compartments of the sarcoplasmic reticulum.
The sarcoplasmic reticulum, which represents a complex sub-compartmentalization of smooth ER, appears as a network of tubules and cisternae, mainly devoted to Ca2+ storage. At least two structurally and functionally distinct subdomains have been identified in the sarcoplasmic reticulum, the longitudinal tubules and the terminal cisternae. However, although these domains have been well described in terms of their morphological and biochemical composition, little is known about the mechanisms directing the organization of sarcoplasmic reticulum domains or about targeting or segregation of specific proteins to specific sub-compartments.
b) molecular mechanism(s) responsible for the interactions between the sarcoplasmic reticulum and myofibrils.
The sarcoplasmic reticulum is organised to form a sleeve-like structure of intracellular membranes organised around each myofibril. In this context, specialised subdomains of the sarcoplasmic reticulum are precisely aligned with respect to specific regions of the sarcomere/contractile apparatus in a structural arrangement that reflects the optimal positioning of Ca2+ release sites with respect to their target: the myofilaments. We have recently discovered that ank1.5, a small splice variant of the ank1 gene localised on the sarcoplasmic reticulum membrane, is capable of interacting with the C-terminus of Obscurin, a giant sarcomeric protein present in the myofibrils. The interaction between ank1.5 and Obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. We are currently extending these studies to identify other components necessary for the assembly of these structures.
c) confocal and epifluorescence microscopy techniques ( FRAP and FRET) to study protein dynamics and interactions in the sarcoplasmic reticulum.
Most of the projects carried out in our lab are based on sophisticated methods for understanding localization and dynamic of proteins of interest in living cells. Fluorescent chimeras of proteins of interest are expressed in cultured myoblasts and observed through live imaging. These techniques take advantage of both laser confocal and epifluorescence microscopy using recently developed protocols such as FRAP (fluorescence microscopy after photobleaching), FRET (fluorescence resonance energy transfer) and time lapse imaging. These techniques allow us to follow proteins of interest through myoblast differentiation until they reach their spatial and functional status in highly differentiated cells. FRAP measurements, which measure the level of mobility of specific proteins, may provide indirect evidence on the possible association of the protein being analysed with relatively stable structures of the cell. FRET analyses, on the other hand, can be used to verify interactions between proteins and within protein complexes.
d) in silico and “wet” cloning of novel muscle-specific genes.
In addition to study known proteins of the sarcoplasmic reticulum, we are working to the identification of novel muscle specific proteins involved in the organization of this organelle. Recent results from our laboratory support the notion that the intracellular organization of the sarcoplasmic reticulum and of of its subcellular domains with respect to the sarcomere is the result of a series of changes which can be followed during muscle development in vivo as well as during differentiation of muscle cells in vitro. To identify and characterise genes encoding proteins involved in this process we are following two complementary strategies: 1) screening of available microarray data sets looking for genes expressed predominantly in muscle tissues, but of unknown function; 2) molecular cloning by Suppression Subtractive Hybridization (SSH) technique to compare gene expression between cells at different levels of organization of the sarcoplasmic reticulum. Selected genes are being characterized with respect to their localization in muscle cells by confocal fluorescence microscopy and by RNA interference (RNAi) in order to verify their function.
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Mechanisms that regulate proliferative and differentiative potentials of human adult stem cells.
Research on adult stem cells is attracting increasing interest in light of the many biological questions that are linked to stem cells in terms of basic research, but also because stem cells hold great promise for the development of new therapies for a variety of disorders for which a cure is not yet available. Since 2004 our group is participating in the Center for Stem Cell Research of the University of Siena, where our research is currently focused on two main projects:
1) Characterisation of human mesenchymal stem cells from adult human tissues.
2) Studies on circulating Endothelial Precursors Cells (EPCs) and their role in different pathological conditions.
a) Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are multipotent adult stem cells with the ability to differentiate into several cell types including adipose, bone and cartilage cells and represent good candidates for cellular therapy and regenerative medicine. The molecular mechanisms that regulate proliferation and differentiation of MSCs are not known. We are interested in elucidating these mechanisms and to compare the characteristics of the mesenchymal stem cells that we have derived from different sources. We also want to verify the regenerative capacity of these cells by using animal models of pathologies such as cardiac infarction and skeletal muscle injury.
Although MSCs were originally isolated from bone marrow, similar populations have been reported in other tissues. In our lab, we have isolated and expanded different populations of MSCs from adipose, cardiac and pancreatic tissues and from umbilical cord blood. The phenotype of these cells has been characterised at the level of cell surface antigen expression by FACS analysis and of gene expression profile by RT-PCR. The potential of these cells to differentiate in different cell types, including adipose, cartilage, bone, skeletal and cardiac muscle cells is being tested in a comparative way to verify whether MSCs from different tissues display similar or different capabilities in their differentiative potentials. Molecular analysis will be performed to verify whether these cells can be distinguished in terms of gene expression aiming to define the molecular pathways that govern proliferation and differentiation in adult human stem cells.
b) Endothelial precursor cells in the pathogenesis of disease states
Endothelial Progenitor Cells (EPCs) are one example of stem cells present in adults. EPCs are a subtype of circulating, bone marrow derived cells with properties similar to those of embryonic angioblasts contained in peripheral blood of adults. These precursors have the potential to differentiate into mature endothelial cells. A feature of EPCs is their ability to form clusters or colony forming units (CFU’s). The influence of pathological conditions on the number of circulating EPCs has been reported in the literature for patients with risk factors for ischemic cardiovascular disease and other pathologies. Again, in spite of the interest on these cells, they are still poorly characterised and relatively poor information is available. The aim of the current project is to screen a cohort of patients with various pathological conditions and healthy controls using a number of cell surface antigens potentially expressed on stem cells with a view to better define the EPC populations circulating in normal and pathological conditions. The techniques applied in this project include fluorescence-activated cell sorter (FACS) for the analysis of peripheral blood mononucleated cells, cell culture and immuno-fluorescence techniques. Molecular techniques are also being applied to isolated populations of EPC to characterise their profile of gene expression.
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