The Actin Nucleator Cobl Is Critical for Centriolar Positioning, Postnatal Planar Cell Polarity Refinement, and Function of the Cochlea.

Proper cochlear hair cell array development and sensory apparatus positioning are achieved by planar cell polarity signaling. Effectors executing proper tissue development and maturation programs are largely unknown. We show that the actin nucleator Cobl is an important effector in postnatal refinement and maintenance of planar cell polarity. During the critical time of hearing onset, these polarity defects coincided with reduced F-actin beneath the sensory apparatus and with premature kinocilium retraction. These defects were accompanied by organizational defects of the pericentriolar scaffold that coincided with basal body and centriolar mispositionings. Importantly, the pericentriolar defects observed in Cobl KO mice were demonstrated to be actin polymerization dependent and calcium/calmodulin signaling dependent. Because Cobl KO phenotypes manifested postnatally, planar cell polarity is not solely an important developmental process. The Cobl-dependent planar cell polarity maintenance and refinement processes we describe here seem critical for hearing, as Cobl KO mice show deficient cochlear amplification.

Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination.

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

Ciliated sensory hair cell formation and function require the F-BAR protein syndapin I and the WH2 domain-based actin nucleator Cobl.

During development, general body plan information must be translated into distinct morphologies of individual cells. Shaping cells is thought to involve cortical cytoskeletal components and Bin-Amphiphysin-Rvs167 (BAR) superfamily proteins. We therefore conducted comprehensive side-by-side loss-of-function studies of zebrafish orthologs of the F-BAR protein syndapin I and the actin nucleator Cobl. Zebrafish syndapin I associates with Cobl. The loss-of-function phenotypes of these proteins were remarkably similar and suggested a common function. Both cobl- and syndapin I-morphant fish showed severe swimming and balance-keeping defects, reflecting an impaired organization and function of the lateral line organ. Their lateral line organs lacked several neuromasts and showed an impaired functionality of the sensory hair cells within the neuromasts. Scanning electron microscopy revealed that sensory hair cells of both cobl- and syndapin I-morphant animals showed defects in the formation of both microtubule-dependent kinocilia and F-actin-rich stereocilia. Consistent with the kinocilia defects in sensory hair cells, body length was shortened and the development of body laterality, a process depending on motile cilia, was also impaired. Interestingly, Cobl and syndapin I both localized to the base of forming cilia. Rescue experiments demonstrated that proper formation of ciliated sensory hair cell rosettes relied on Cobl's syndapin I-binding Cobl homology domain, the actin-nucleating C-terminus of Cobl and the membrane curvature-inducing F-BAR domain of syndapin I. Our data thus suggest that the formation of distinct types of ciliary structures relies on membrane topology-modulating mechanisms that are based on F-BAR domain functions and on complex formation of syndapin I with the actin nucleator Cobl.

Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I.

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.

Cathepsin K deficiency partially inhibits, but does not prevent, bone destruction in human tumor necrosis factor-transgenic mice.

OBJECTIVE: Cathepsin K is believed to have an eminent role in the pathologic resorption of bone. However, several studies have shown that other proteinases also participate in this process. In order to clarify the contribution of cathepsin K to the destruction of arthritic bone, we applied the human tumor necrosis factor (hTNF)-transgenic mouse model, in which severe polyarthritis characterized by strong osteoclast-mediated bone destruction develops spontaneously. METHODS: Arthritis was evaluated in hTNF-transgenic mice that were either wild-type for cathepsin K (CK(+/+)), heterozygous for cathepsin K (CK(+/-)), or deficient in cathepsin K (CK(-/-)). Arthritic knee joints were prepared for standard histologic assessment aimed at establishing a semiquantitative score for joint destruction and quantification of the area of bone erosion. Additionally, microfocal computed tomography was performed to visualize bone destruction in mice with the different CK genotypes. CK(+/+) and CK(-/-) osteoclasts were generated in vitro, and their proteinase expression profiles were compared by complementary DNA array analysis, real-time polymerase chain reaction, and activity assays. RESULTS: Although the area of bone erosion was significantly reduced in hTNF-transgenic CK(-/-) mice, the absence of cathepsin K did not completely protect against inflammatory bone lesions. Several matrix metalloproteinases (MMPs) and cathepsins were expressed by in vitro-generated CK(-/-) osteoclasts, without marked differences from CK(+/+) osteoclasts. MMP activity was detected in CK(-/-) osteoclasts, and MMP-14 was localized by immunohistochemistry in inflammatory bone erosions in hTNF-transgenic CK(-/-) mice, suggesting MMPs as potential contributors to bone destruction. Additionally, we detected a reduction in osteoclast formation in cathepsin K-deficient mice, both in vitro and in vivo. CONCLUSION: The results of our experiments raise doubts about a crucial role of cathepsin K in arthritic bone destruction.

Optimized folding and activation of recombinant procathepsin L and S produced in Escherichia coli.

Large scale production of the recombinant human cathepsins L and S was optimized. Final purity was nearly 100%, yield 65% and 41%, respectively. Cost-effective expression in Escherichia coli, inclusion body purification and solubilization followed modified standard protocols. Most contribution to the remarkable increase in over-all efficiency came from the subsequent steps: folding by dilution, selective HIC-capturing of the folded proenzymes, and auto-activation. The effort to optimize the process parameters for folding and activation was greatly reduced by central composite fractional factorial experimental design, considering curved responses as well as factor interactions. Theoretical and practical features of this powerful tool for experimental design are given. Yield in procathepsin S folding could be further increased by addition of an excess of its own native propeptide with known intramolecular chaperone function. This corroborates literature data on proenzyme folding and is broadly discussed in the light of the lower conformational stability of the prodomain compared to the catalytic unit. Auto-activation kinetics was largely different between the two related proenzymes; from its time course contribution of uni- and bimolecular events in proregion hydrolysis and rate constants were estimated.

An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube format.

BACKGROUND: Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes. Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures. RESULTS: In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well. CONCLUSION: As the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.