Membrane remodelling triggers maturation of excitation–contraction coupling in 3D-shaped human-induced pluripotent stem cell-derived cardiomyocytes

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca$^{2+}$-dependent excitation–contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca$^{2+}$ channels critical for EC coupling in close proximity, the L-type Ca$^{2+}$ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca$^{2+}$-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca$^{2+}$ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches

Enhancer-associated H3K4 methylation safeguards in vitro germline competence.

Funder: Studienstiftung des Deutschen VolkesGermline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions

Untersuchungen zur Dimerisierung von Somatostatin-Rezeptoren mit anderen G-Protein-gekoppelten Rezeptoren

Heterodimerization, G-Protein-Coupled Receptors, somatostatin receptor, internalization, oligomerization, dimerization, homodimerization, GPCR, desensitization, immunoprecipitationMagdeburg, Univ., Fak. für Naturwiss., Diss., 2004von Magdalena Laugsc

Generation of inducible immortalized dendritic cells with proper immune function in vitro and in vivo.

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1β, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4(+) T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells

Characterization of cytoplasmic SMC3 protein.

<p>(<b>A</b>) Expression of mRNA of Smc1, Smc3 and Rad21 72 h after esiSMC1 treatment, compared to control (esiEGFP), was examined by real-time RT-PCR. Relative quantification of gene expression was achieved by normalization to ß-actin, (n = 3). (<b>B</b>) Effect of SMC1 knockdown on RAD21 protein in total RIPA extracts as monitored by IB with anti-Rad21 antibody 72 h post esiSMC1 or esiEGFP transfection. Bottom: quantification of IB from three independent experiments. (<b>C</b>) Sequential IP. IP #1 from nuclear and cytoplasmic extracts from esiSMC1-treated and control cells. The supernatant was used for IP#2 with anti-SMC3 antibody. Eluates were analyzed by IB using anti-RAD21, -SMC3, and -SMC1 antibodies. (<b>D</b>) Nuclear export was inhibited 70 h after esiSMC1 or control treatment by addition of LMB to a final concentration of 5 ng/mL for another 2 h. The localization of SMC3 (in red, top half) was examined by immunofluorescence. NFκB (in red, bottom half) was used to confirm the LMB effect. DNA was visualized by DAPI (in blue). (<b>E</b>) Immunoprecipitation of SMC3 from cytoplasm and nuclear extracts after LMB inhibition was performed and eluates examined by silver staining. Arrows indicate the positions of SMC1, SMC3 and RAD21. The asterisk indicates an unspecific band as specified by mass spectrometric analysis.</p

Imbalance of SMC1 and SMC3 Cohesins Causes Specific and Distinct Effects

<div><p>SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.</p></div

Transient down-regulation of endogenous human SMC1 in HeLa cells using specific esiRNA or siRNA impairs the nuclear localization of SMC3.

<p>(<b>A</b>) RIPA total cell extracts were prepared 72 h after treatment with three different concentrations of esiSMC1 and examined by IB using anti-SMC1 antibody. Mock transfected cells were used as negative control. The membrane was reprobed with anti-SMC3 antibody to confirm the specificity of esiSMC1 and equal loading. The percentages of SMC1 protein levels, normalized to SMC3 protein levels with respect to mock control set at 100%, are indicated. (<b>B</b>) Kinetics of recovery of SMC1 expression after treatment of cells with 750 ng/mL of esiSMC1 was analyzed by IB as described in A. (<b>C</b>) Quantification of SMC1 and SMC3 in RIPA total extracts of cells treated with 750 ng/mL of esiRNA and collected 72 h post transfection. Average of six independent experiments is shown. (<b>D</b>) IF microscopic analysis of SMC1 knockdown 72 h post esiRNA transfection by anti-SMC1 staining in red and DAPI in blue. Specific esiRNA against EGFP (esiEGFP) was used as a control. (<b>E</b>) IF microscopic analyses of esiSMC1- or esiEGFP-treated cells (72 h) using anti-SMC3 (red) and DAPI (blue). (<b>F</b>) Cytoplasmic and nuclear extracts from esiSMC1 or control treated cells were analyzed by IB using anti-SMC3. The membrane was reprobed with anti-SMC1. Anti-ß tubulin and Topo II antibodies were used to determine the purity of nuclear and cytoplasm extracts. (<b>G</b>) Quantification of results from four independent experiments that were performed as described in F. (<b>H</b>) Time course of SMC3 localization upon treatment of cells with siSMC1 (#1) as visualized by IF microscopy using anti-SMC3 (in red) and DAPI (in blue).</p

FRAP experiments using EGFP-msSMC1 (A) or EGFP-msSMC3 (B) as bleach substrates.

<p>The mobile fraction (recovery), the intensity of the EGFP-tagged protein signal (given as mean grey values), and tau (τ) as a value inversely correlating with mobility are shown; ns  =  non significant; p-values are shown within each graph.</p

Human hair follicles are an extrarenal source and a nonhematopoietic target of erythropoietin

ABSTRACT Erythropoietin primarily serves as an essential growth factor for erythrocyte precursor cells. However, there is increasing evidence that erythropoietin (EPO)/EPO receptor (EPO‐R) signaling operates as a potential tissue‐protective system outside the bone marrow. Arguing that growing hair follicles (HF) are among the most rapidly proliferating tissues, we have here explored whether human HFs are sources of EPO and targets of EPO‐R‐mediated signaling. Human scalp skin and microdissected HFs were assessed for EPO and EPO‐R expression, and the effects of EPO on organ‐cultured HFs were assessed in the presence/ absence of a classical apoptosis‐inducing chemothera‐peutic agent. Here, we show that human scalp HFs express EPO on the mRNA and protein level in situ, up‐regulate EPO transcription under hypoxic conditions, and express transcripts for EPO‐R and the EPO‐stimulatory transcriptional cofactor hypoxia‐inducible factor‐1α. Although EPO does not significantly alter human hair growth in vitro, it significantly down‐regulates chemotherapy‐induced intrafollicular apoptosis and changes the gene expression program of the HFs. The current study points to intriguing targets of EPO beyond the erythropoietic system: human HFs are an extrarenal site of EPO production and an extrahema‐topoietic site of EPO‐R expression. They may recruit EPO/EPO‐R signaling e.g., for modulating HF apopto‐sis under conditions of hypoxia and chemotherapy‐induced stress.—Bodó, E., Kromminga, A., Funk, W., Laugsch, M., Duske, U., Jelkmann, W., Paus, R. Human hair follicles are an extrarenal source and a non‐hematopoietic target of erythropoietin. FASEB J. 21, 3346–3354 (2007

Two-species system: knockdown of human SMC3 by siRNA and rescue by stably expressed EGFP-msSMC3.

<p>(<b>A</b>) Cells stably transfected with EGFP-msSMC3 were analyzed by IF microscopy. (<b>B</b>) Total cell (RIPA), cytoplasmic and nuclear extracts from cells stably expressing EGFP-msSMC3 collected 72 h after treatment with siSMC1 or siSMC3 were analyzed by IB using anti-EGFP, -SMC3 and -SMC1 antibodies. RIPA buffer was used for the final extraction step after the nuclear extraction with 250 mM ammonium sulfate. Topo II and Karyopherin ß1 were used as a loading control. Relative protein levels are shown at the bottom (representative of 3 experiments).</p