[Show abstract][Hide abstract] ABSTRACT: Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon-mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.
[Show abstract][Hide abstract] ABSTRACT: Rationale. Critical illness myopathy (CIM) has no known cause and no treatment. Immobilization and impaired glucose metabolism are implicated. Objectives. We assessed signal transduction in skeletal muscle of patients at risk for CIM. We also investigated the effects of evoked muscle contraction (ISRCTN77569430). Methods. Prospective observational and interventional pilot study. We screened 874 mechanically ventilated patients with sepsis-related organ-failure assessment score ≥ 8 for three consecutive days in the first five days of ICU. 30 patients at risk for CIM underwent euglycemic-hyperinsulinemic clamp, muscle microdialysis studies, and muscle biopsies. Controls were healthy. In five additional patients at risk for CIM, we performed corresponding analyses after 12-day, daily, unilateral electrical muscle stimulation (EMS) with the contralateral leg as control. Measurements. We performed successive muscle biopsies, assessed systemic insulin sensitivity and signal transduction pathways of glucose utilization at mRNA and protein level and glucose transporter-4 (GLUT4) localization in skeletal muscle tissue. Main results. Skeletal muscle GLUT4 was trapped at perinuclear spaces, most pronounced in CIM patients, but resided at the sarcolemma in controls. Glucose metabolism was not stimulated during euglycemic-hyperinsulinergic clamp. Insulin signal transduction was competent up to p-Akt activation; however, p-AMPK was not detectable in CIM muscle. EMS increased p-AMPK, repositioned GLUT4, locally improved glucose metabolism, and prevented type-2 fiber atrophy. Conclusions. Insufficient GLUT4 translocation results in decreased glucose supply in CIM patients. Failed AMPK activation is involved. Evoked muscle contraction may prevent muscle-specific AMPK failure, restore GLUT4 disposition, and diminish protein breakdown.
American Journal of Respiratory and Critical Care Medicine 12/2012; 187(4). DOI:10.1164/rccm.201209-1649OC · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca(2+)dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients' myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.
PLoS ONE 11/2012; 7(11):e49603. DOI:10.1371/journal.pone.0049603 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle is continually subjected to microinjuries that must be repaired to maintain structure and function. Fluorescent dye influx after laser injury of muscle fibers is a commonly used assay to study membrane repair. This approach reveals that initial resealing only takes a few seconds. However, by this method the process of membrane repair can only be studied in part and is therefore poorly understood. We investigated membrane repair by visualizing endogenous and GFP-tagged repair proteins after laser wounding. We demonstrate that membrane repair and remodeling after injury is not a quick event but requires more than 20 min. The endogenous repair protein dysferlin becomes visible at the injury site after 20 seconds but accumulates further for at least 30 min. Annexin A1 and F-actin are also enriched at the wounding area. We identified a new participant in the membrane repair process, the ATPase EHD2. We show, that EHD2, but not EHD1 or mutant EHD2, accumulates at the site of injury in human myotubes and at a peculiar structure that develops during membrane remodeling, the repair dome. In conclusion, we established an approach to visualize membrane repair that allows a new understanding of the spatial and temporal events involved.
[Show abstract][Hide abstract] ABSTRACT: Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well as to test therapeutic strategies at the cellular level.
[Show abstract][Hide abstract] ABSTRACT: The treatment of cells with histone deacetylase inhibitors (HDACi) was reported to reveal the acetylation of STAT1 at lysine
410 and lysine 413 (O. H. Krämer et al., Genes Dev. 20:473–485, 2006). STAT1 acetylation was proposed to regulate apoptosis by facilitating binding to NF-κB and to control immune
responses by suppressing STAT1 tyrosine phosphorylation, suggesting that STAT1 acetylation is a central mechanism by which
histone deacetylase inhibitors ameliorate inflammatory diseases (O. H. Krämer et al., Genes Dev. 23:223–235, 2009). Here, we show that the inhibition of deacetylases had no bearing on STAT1 acetylation and did not diminish
STAT1 tyrosine phosphorylation. The glutamine mutation of the alleged acetylation sites, claimed to mimic acetylated STAT1,
similarly did not diminish the tyrosine phosphorylation of STAT1 but precluded its DNA binding and nuclear import. The defective
transcription activity of this mutant therefore cannot be attributed to STAT1 acetylation but rather to the inactivation of
the STAT1 DNA binding domain and its nuclear import signal. Experiments with respective cDNAs provided by the authors of the
studies mentioned above confirmed the results reported here, further questioning the validity of the previous data. We conclude
that the effects and potential clinical benefits associated with histone deacetylase inhibition cannot be explained by promoting
the acetylation of STAT1 at lysines 410 and 413.
[Show abstract][Hide abstract] ABSTRACT: The biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments revealed that fully tyrosine-phosphorylated STAT dimers polymerize via Tyr(P)-Src homology 2 domain interactions and assemble into paracrystalline arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs from dephosphorylation. Activated STAT3 forms such paracrystals in acute phase liver cells. Activated STAT1, in contrast, does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is due to the unique ability of STAT1 among the STATs to conjugate to small ubiquitin-like modifier (SUMO). Sumoylation had one direct effect; it obstructed proximal tyrosine phosphorylation, which led to semiphosphorylated STAT dimers. These competed with their fully phosphorylated counterparts and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation, profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study thus identifies polymerization of activated STAT transcription factors as a positive regulatory mechanism in cytokine signaling. It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors and reconciles the conflicting results as to the role of SUMO modification in STAT1 functioning. We present a generally applicable system in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors.
[Show abstract][Hide abstract] ABSTRACT: The biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to
elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments
uncovered that fully tyrosine-phosphorylated STAT dimers polymerize via pTyr-SH2 domain interactions, and assemble into paracrystalline
arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs
from dephosphorylation. Activated STAT3 forms such paracrystals in acute-phase liver cells. Activated STAT1, in contrast,
does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is
due to STAT1′s unique ability among the STATs to SUMO conjugate. Sumoylation had one direct effect: it obstructed proximal
tyrosine phosphorylation, which lead to semi-phosphorylated STAT dimers. These competed with their fully phosphorylated counterparts
and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation,
profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study
thus identifies polymerization of activated STAT transcription factors as a positive-regulatory mechanism in cytokine signaling.
It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors, and reconciles
the conflicting results as to the role of SUMO modification for STAT1 functioning. We present a generally applicable system
in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO
partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors.
Journal of Biological Chemistry 03/2011; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The AHNAK scaffold PDZ-protein family is implicated in various cellular processes including membrane repair; however, AHNAK function and subcellular localization in skeletal muscle are unclear. We used specific AHNAK1 and AHNAK2 antibodies to analyzed the detailed localization of both proteins in mouse skeletal muscle. Co-localization of AHNAK1 and AHNAK2 with vinculin clearly demonstrates that both proteins are components of the costameric network. In contrast, no AHNAK expression was detected in the T-tubule system. A laser wounding assay with AHNAK1-deficient fibers suggests that AHNAK1 is not involved in membrane repair. Using atomic force microscopy (AFM), we observed a significantly higher transverse stiffness of AHNAK1⁻/⁻ fibers. These findings suggest novel functions of AHNAK proteins in skeletal muscle.
Biochemical and Biophysical Research Communications 10/2010; 401(1):143-8. DOI:10.1016/j.bbrc.2010.09.030 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the nuclear envelope. They are distinguished by their ability to trigger the nucleocytoplasmic redistribution of their antigen. What determines these apparently contradictory outcomes has not been explored. We studied a stationary STAT1 antibody and a translocating importin-beta antibody. The stationary phenotype resulted from the inhibition of carrier-independent transport. This was not due to crosslinking or precipitation of antigen, because the antigen-antibody complex remained highly mobile. Rather, decoration with stationary antibody precluded actual nuclear pore passage of antigen. In addition, both antibodies inhibited the carrier-dependent translocation via importin-alpha, but by diverse mechanisms. The translocating antibody blocked the association with importin-alpha, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-alpha binding step. We identified a stationary antibody to green-fluorescent protein (GFP) and probed the translocation of GFP fusions of STAT1, thyroid hormone receptor and histones, demonstrating general application of this approach. Our results provide an experimental rationale for the use of antibodies as unique tools for dissecting protein nuclear translocation. As the microinjection of stationary antibodies extends to analyses of native proteins, this method can complement and validate results obtained with fluorescent-labeled derivatives.
Cytometry Part A 12/2008; 73A(12):1128-40. DOI:10.1002/cyto.a.20635 · 2.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytokine-dependent gene activation critically depends upon the tyrosine phosphorylation (activation) of STAT transcription factors at membrane-bound cytokine receptors. The extent of STAT activation and hence the specificity of signaling is primarily determined by structural complementarity between the SH2 domain of the STATs and the tyrosine-phosphorylated receptor chains. Here, we identified constitutive nucleocytoplasmic shuttling as another mechanism that controls the differential activation of STAT transcription factors. Our analysis of nucleocytoplasmic cycling of STAT1 revealed that the expression of the alternatively spliced transactivation domain and its signal-dependent serine phosphorylation maximized the rate of nuclear export. Export modulation occurred independently of retention factors or the export receptor CRM1, and was observed both before and during stimulation of cells with cytokines. Our data indicated a dual role for the transactivation domain. It enhanced the nuclear retention of activated STAT1, but had the opposite effect on inactivated molecules. Accordingly, and despite their identical receptor recognition, the STAT1 splice variants differed strongly in the amplitude of tyrosine phosphorylation and in the duration of the cytokine signal. Thus, regulated nuclear export determined the cytokine sensitivity of the shuttling STAT1 transcription factors by controlling their availability at the receptor kinase complex.
[Show abstract][Hide abstract] ABSTRACT: Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH(2)- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.
The Journal of Cell Biology 07/2004; 165(6):823-33. DOI:10.1083/jcb.200403057 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytokine-dependent gene transcription greatly depends on the tyrosine phosphorylation ("activation") of Stat proteins at the cell membrane. This rapidly leads to their accumulation in the nucleus by an unknown mechanism. We performed microinjections of recombinant Stat1 protein to show that nuclear accumulation of phosphorylated Stat1 can occur without cytokine stimulation of cells. Microinjection of Stat1 antibody and treatment of cells with kinase or phosphatase inhibitors revealed that nuclear accumulation is a highly dynamic process sustained by Stat1 nucleocytoplasmic cycling and continuous kinase activity. By characterizing nuclear accumulation mutants, it is demonstrated that nuclear import and nuclear retention are two separate steps leading up to nuclear accumulation, with nonspecific DNA binding of activated Stat1 being sufficient for nuclear retention. Critical for nuclear buildup of Stat1 and the subsequent nuclear export is the point of time of tyrosine dephosphorylation, because our data indicate that activated Stat1 is incapable of leaving the nucleus and requires dephosphorylation to do so. It is demonstrated that the inactivation of Stat1 is controlled by its exchange reaction with DNA, whereby DNA binding protects Stat1 from dephosphorylation in a sequence-specific manner. Thus, during nuclear accumulation, a surprisingly simple mechanism integrates central aspects of cytokine-dependent gene regulation, for example, receptor monitoring, promoter occupancy, and transcription factor inactivation.
Genes & Development 09/2003; 17(16):1992-2005. DOI:10.1101/gad.268003 · 10.80 Impact Factor