The cyclin‐dependent kinase inhibitor p21CIP1/WAF1 is a regulatory factor of the cell cycle. Its transcriptional activation and protein stability are tightly controlled by several distinct mechanisms. S100A11 is a member of the S100 family of Ca2+‐binding proteins involved in several biological processes, including cell cycle progression and signal transduction. In the present study, we show that down‐regulation of S100A11 results in the reduction of p21 protein in human HaCaT keratinocytes. It appears that a ubiquitin‐independent proteasomal degradation process is involved in p21 degradation in S100A11 down‐regulated cells. The application of a proteasome inhibitor stabilized p21 protein in these cells. Analysis of distinct signal transduction pathways revealed a disturbed phosphatidylinositol‐3‐kinase/Akt pathway after S100A11 knockdown. We determined that the glycogen synthase kinase‐3, which is negatively regulated by phosphatidylinositol 3‐kinase/Akt, was activated in cells possessing knocked‐down S100A11 and appears to be involved in p21 protein destabilization. The application of a specific inhibitor of glycogen synthase kinase 3 resulted in an increase of the p21 protein level in S100A11 down‐regulated HaCaT cells. Glycogen synthase kinase 3 is able to phosphorylate p21 at T57, which induces p21 proteasomal turnover. Mutation of the glycogen synthase kinase 3 site threonine 57 into alanine (T57A) stabilizes p21 in HaCaT cells lacking S100A11. Beside decreased p21 protein, down‐regulation of S100A11 triggered the induction of apoptosis in HaCaT cells. These observations suggest that S100A11 is involved in the maintenance of p21 protein stability and appears to function as an inhibitor of apoptosis in human HaCaT keratinocyte cells. Thus, the data shed light on a novel pathway regulating p21 protein stability.

Chain length, size, composition, surface charge, and other properties of polymeric materials affect their recognition and uptake by cells and must be optimized to deliver polymers selectively to their target. However, it is often not possible to precisely modify selected properties without changing other parameters. To overcome these difficulties, well-defined poly(pentafluorostyrene)-based polymers are prepared that can be grafted via thiol/para-fluorine "click" reaction with 1-thio-β-D-glucose and 1-thio-β-D-galactose. Fluorescence microscopy and flow cytometry show that nanoparticles are taken up by HepG2 cells to a higher degree than the respective water-soluble polymers, and that internalization of both galactosylated homo- and nanoprecipitated block copolymers is enhanced.

Cytochrome C oxidase and FoF1-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent cryo-tomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.

No abstract

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca(2+), a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.

Knowledge about precise numbers of specific molecules is necessary for understanding and verification of biological pathways. The RAD51 protein is central in the repair of DNA doublestrand breaks (DSBs) by homologous recombination repair and understanding its role in cellular pathways is crucial to design mechanistic DNA repair models. Here, we determined the number of RAD51 molecules in several human cell lines including primary fibroblasts. We showed that between 20000 to 100000 of RAD51 molecules are available per human cell that theoretically can be used for simultaneously loading at least 7 DSBs. Interestingly, the amount of RAD51 molecules does not significantly change after the induction of DNA damage using bleomycin or γirradiation in cells but an accumulation of RAD51 on the chromatin occurs. Furthermore, we generated an EGFP-RAD51 fusion under the control of HSV thymidine kinase promoter sequences yielding moderate protein expression levels comparable to endogenously expressed RAD51. Initial characterizations suggest that these low levels of ectopically expressed RAD51 are compatible with cell cycle progression of human cells. Hence, we provide parameters for the quantitative understanding and modeling of RAD51-involving processes.

No abstract

No abstract