Smad2/3 potentiate cell identity conversions with master transcription factors

The exogenous expression of master transcription factors (TFs) to drive cell identity changes is an exciting and powerful approach to cell and tissue engineering. Yet, the generation of desired cell types is often plagued by inefficiency and inability to produce mature cell types. Through investigations of the molecular mechanisms of induced pluripotent stem cell (iPSC) generation, I discovered that expression of constitutively active Smad2/3 (Smad2CA/3CA), together with the Yamanaka factors, could dramatically improve the efficiency of reprogramming. Mechanistically, SMAD3 interacted with both co-activators and reprogramming factors, bridging their interaction during reprogramming. Because SMAD2/3 interact with a multitude of master TFs in different cell types, I tested the conversions of B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. Remarkably, each conversion system was markedly enhanced when the master TFs were co-expressed with Smad3CA. These results revealed the existence of shared molecular mechanisms underlying diverse TF-mediated cellular conversions, and demonstrated SMAD2/3 as a widely applicable cofactor that potentiates the generation of diverse cell types with profound efficiency and maturity

Shigella flexneri utilize the spectrin cytoskeleton during invasion and comet tail generation

<p>Abstract</p> <p>Background</p> <p>The spectrin cytoskeleton is emerging as an important host cell target of enteric bacterial pathogens. Recent studies have identified a crucial role for spectrin and its associated proteins during key pathogenic processes of <it>Listeria monocytogenes </it>and <it>Salmonella </it>Typhimurium infections. Here we investigate the involvement of spectrin cytoskeletal components during the pathogenesis of the invasive pathogen <it>Shigella flexneri.</it></p> <p>Results</p> <p>Immunofluorescent microscopy reveals that protein 4.1 (p4.1), but not adducin or spectrin, is robustly recruited to sites of <it>S. flexneri </it>membrane ruffling during epithelial cell invasion. Through siRNA-mediated knockdowns, we identify an important role for spectrin and the associated proteins adducin and p4.1 during <it>S. flexneri </it>invasion. Following internalization, all three proteins are recruited to the internalized bacteria, however upon generation of actin-rich comet tails, we observed spectrin recruitment to those structures in the absence of adducin or p4.1.</p> <p>Conclusion</p> <p>These findings highlight the importance of the spectrin cytoskeletal network during <it>S. flexneri </it>pathogenesis and further demonstrate that pathogenic events that were once thought to exclusively recruit the actin cytoskeletal system require additional cytoskeletal networks.</p

Constitutively active Smad2/3 are broad scope potentiators of transcription factor-mediated cellular reprogramming

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering. Ruetz et al. show that constitutively active SMAD2/3 has a surprising ability to boost the efficiency of cell reprogramming both to iPSCs and across lineages and may therefore be a general factor that can enhance transcription-factor-mediated reprogramming in a variety of contexts

The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress

Characterizing the role of the spectrin cytoskeleton during adherent and invasive bacterial pathogenesis

The enteric pathogens, enteropathogenic Escherichia coli (EPEC), Salmonella Typhimurium and Listeria monocytogenes cause millions of infections worldwide each year. A shared host cell target of these microbes is the cytoskeleton, which is reorganized as part of the infectious process. Because the hijacking of the cytoskeleton is a pre-requisite for disease, I hypothesized that the spectrin cytoskeleton would be targeted by these pathogens. I investigated three major components of the spectrin cytoskeleton: spectrin, adducin and protein 4.1. Using immunolocalization techniques, I identified spectrin cytoskeletal components recruited to EPEC pedestals, S. Typhimurium membrane invasion ruffles and Salmonella containing vacuoles (SCVs), as well as L. monocytogenes actin phagocytic cups and sites of initial comet tail formation. When any of the spectrin cytoskeletal proteins were knocked down, pathogenesis of each organism was severely attenuated. These findings reveal a novel host cell cytoskeletal network that is crucial for both adherent and invasive bacterial disease

Constitutively active SMAD2/3 are broad-scope potentiators of transcription-factor-mediated cellular reprogramming

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.This work was supported by the ERC (grants ROADTOIPS 261075 to K.K. and BRAINCELL 261063 to S.L. and iN-Brain 309712 to M.P.), the BBSRC (project grant BB/L023474/1 to K.K.), the Anne Rowling Regenerative Neurology Clinic (K.K.), the Swedish Research Council (grant STARGET to S.L. and grants 521-2012-5624 and 521-2013-3347 to M.P.), and the Wellcome Trust (grant WT098051 to K.Y.). T.R., L.T., J.A., and D.F.K. are funded by a Darwin Trust scholarship, a CMVM scholarship, and Principal's Career Development scholarship from the University of Edinburgh, respectively. D.F.K. is supported by the BBSRC (EASTBIO doctoral training partnership). T.V.T. is supported by a Juan de la Cierva postdoctoral fellowship (MINECO, FJCI-2014-22946). B.D.S. was supported by an EMBO long-term fellowship (ALTF 1143-2015). K.K. is an MRC senior non-clinical fellow (MR/N008715/1). M.P. is a New York Stem Cell Foundation Robertson Investigator