From an evolutionary perspective the high level of resistanc

From an evolutionary perspective, the high level of resistance of HSPCs to lentiviruses makes sense, since any major chromosomal vulnerability would have more serious implications than in progeny cells. The high-level expression of SAMHD1 in cultured HSPCs is reminiscent of the one in activated CD4+ T cells (Baldauf et al., 2012). In activated CD4+ T cells, the high expression of SAMHD1 goes along with a high concentration of dNTP and high degree of permissiveness to HIV and HIV-1-based transduction. In HSPCs, we encounter an entirely different situation with a low concentration of dNTPs and a low permissiveness to HIV-1-based lentiviral vectors. Thus, each cellular setting must be examined separately.
Conclusions In summary, HSPCs, in culture, express high levels of SAMHD1. Vpx-mediated decrease of SAMHD1 relieves only marginally the restriction of lentiviral-based transduction. The data imply that other blocks mainly at cell entry are the major limiting step for efficient transduction. Indeed, a recent study showed that the LDL receptor acts as the receptor for VSV-G-pseudotyped particles (Amirache et al., 2014), and lack of it appears to be at the origin of the poor permissiveness of HSPCs to lentiviral transduction. The following are the supplementary data related to this article.
Acknowledgments We thank D. Boden (Tibotec, Belgium) for providing pLen-EF1α-GFP and the NIH AIDS Reagent Program for providing Raltegravir (Cat # 11680; from Merck & Company). We are grateful to N.R. Landau, O.T. Fackler H.M. Baldauf, V. Vongrad, Y.L. Kok and A. Scherrer for advice. The study was supported by the OPO-Foundation and the clinical research focus program “Human Hemato-Lymphatic Diseases” of the University of Zürich (10/2012-10/2015). RFS is supported by the SNF (SSAJRP; IZLSZ3_149100/1). This work was partially supported by US National Institute of Health grant GM104198 (B.K.).
Introduction Cytoskeleton is a cellular scaffolding contained within cytoplasm. It maintains the cell shape, provides mechanical strength, directs locomotion, regulates chromosome separation in mitosis and meiosis and intracellular transport of organelles in cells (Doherty and McMahon, 2008; Van Troys et al., 2008). Actin microfilaments are the major structure of the cytoskeleton. The Fmoc-Leu-OH manufacturer protein exists within cells in either globular/monomer (G-actin) or filamentous (F-actin) forms and thus in highly dynamic transitions of depolymerization and polymerizationin. During depolymerization, polymerized actin (F-actin) is severed/depolymerized and turns to monomer actin (G-actin). G-actin can be recycled, transferred back into filament form by an ADP-to-ATP exchange. ATP-actin becomes available for assembly and polymerization to F-actin (Ono, 2007). The process of assembly and disassembly of actin filaments in cells is regulated by actin depolymerizing factors (ADFs) that in mammals include: Cofilin1 (CFL1, non-muscle Cofilin), Cofilin2 (CFL2, muscle Cofilin), and Destrin (DSTN, also called ADF or Corn1). CFL1 is ubiquitously expressed, while CFL2 is only expressed in muscles. DSTN also exhibits ubiquitous expression, and its levels are about 5% to 10% of CFL1 levels (Vartiainen et al., 2002). Cofilin binds to actin monomers and filaments, causing depolymerization of actin filaments preventing their reassembly (Ghosh et al., 2004). Phosphorylation and dephosphorylation regulate Cofilin’s binding and associating activity with actin (Lappalainen and Drubin, 1997; Maekawa et al., 1999). DSTN is a component protein in microfilaments. It severs actin filaments (F-actin) and binds to G-actin, thereby, sequestering actin monomers and preventing polymerization (Hawkins et al., 1993). During lineage specific differentiation, human stromal (skeletal) stem cells (hMSCs) exhibit significant changes in morphology and actin cytoskeletal organization (McBeath et al., 2004; Yourek et al., 2007; Treiser et al., 2010). For example, during adipocyte differentiation, the cells undergo a morphological change from fibroblastic to spherical cells filled with lipid droplets (Fan et al., 1983). The change in cell shape takes place early in the differentiation process prior to the up-regulation of many adipocyte specific genes and in association with cytoskeletal changes including decreased actin synthesis and actin reorganization (Antras et al., 1989). Altered actin organization influences cytoskeletal tension which has been demonstrated to play a role in adipogenesis in cultured MSCs (McBeath et al., 2004). Similarly, during chondrocyte differentiation of hMSCs, significant changes in cell shape that transform from a fibroblast-like to a circular morphology leading to a significant increase in cell volume (Xu et al., 2008). Similarly, the regulatory role of actin dynamics in chondrocyte differentiation has been supported by a number of experiments. Actin-disrupting compounds, such as cytochalasin D, stimulate chondrogenesis (Loty et al., 1995). In addition, intracellular kinases induced by adhesion signaling from extracellular matrix (ECM) proteins, regulate chondrocyte differentiation through changes in actin cytoskeleton (Woods et al., 2005, 2007; Nurminsky et al., 2007).