Functional Analysis of the Xin-Repeat Protein Family in Cross-striated Muscle

The assembly of the sarcomere of cross-striated muscle involves a plethora of transcription factors, signalling and structural proteins and requires their coordinate interplay in order to construct this uniquely regular structure. Its early upregulation in muscle precursor cells during embryonic development and in activated satellite cells upon skeletal muscle damage render the protein Xin an interesting candidate for being involved in orchestrating muscle morphogenesis. The establishment of the H-2K cell line as a new reliable model system for studying myofibrillogenesis in vitro using immunofluorescence microscopy and protein expression analysis of major structural components of the sarcomere in this work provided a new tool to investigate Xin function during myogenic differentiation. Examining the transcription level revealed that all three Xin isoforms A, B and C are being upregulated upon initiation of differentiation while downregulated as soon as H-2K cells gain their contractility. On the protein level, only the expression of isoforms A and B was doubtlessly identified exhibiting a course related to their transcriptional level. During differentiation, Xin was strongly associated with non-striated myofibrils (NSMF), where it colocalized with its binding partner filamin C, but not with Z-disc precursor structures. Consistently, in contractile myotubes Xin was absent from mature Z-discs but longitudinally connected to immature sarcomeres. In this work, the SH3 domains of nebulin and nebulette were identified as novel ligands of Xin isoforms A and C and peptide scans elucidated for the first time the exact peptide motif and the residues essential for this interaction. Bimolecular fluorescence complementation (BiFC) assays of Xin C and nebulette in embryonic cardiomyocytes selectively demonstrated the potential cellular sites of this interaction since BiFC complexes were exclusively formed along NSMF's in early developmental stages but not at mature Z-discs. This finding was corroborated by localization studies of nebulin and Xin in H-2K cells as colocalization was only observed at specific sites of NSMF's substantiating the developmental regulation of this interaction. Identification of the protein LIMCH1 as the first binding partner exclusively interacting with the largest Xin isoform A provided first insights into isoform-specific functions. Yeast two-hybrid and biochemical studies mapped the binding interface to the C-terminal LIM domain of LIMCH1 and impressively showed its specificity. Investigation on the mRNA and protein level during myofibrillogenesis of H-2K cells displayed that LIMCH1 is also upregulated at the onset of differentiation constantly increasing until the final stage pointing to an interaction during that process. In rat skeletal muscle tissue and H-2K cells an additional larger LIMCH1 isoform (LIMCH1 muscle) was detected which presumably contains the LIM domain and functional coiled-coil motifs like the other isoform LIMCH1 FL. BiFC experiments in A7r5 cells using fragments of LIMCH1 and Xin might imply that LIMCH1 is capable of restricting the site of their interaction. The Xin-Repeat protein family in human comprises two members, Xin and the larger Xirp2. Since both share a similar domain layout and the ability to bind actin filaments and Ena/VASP proteins, dissection of complementary and exclusive binding properties suggests discrimination of their function. While Xirp2, similar to Xin, could be shown to be a filamin C-specific binding protein which can form multimers and also interacts with the SH3 domain of nebulin and nebulette via a consensus motif, the interaction with α-actinin is an exclusive quality of the Xirp2 repeat region. Furthermore, this study clearly demonstrated that human Xirp2 is upregulated during myogenic differentiation and, in contrast to Xin, a constituent of the mature Z-disc. Immunofluorescence studies using antibodies raised against different epitopes of Xirp2 provided a first clue to the spatial orientation of Xirp2 within the Z-disc thereby determining the arrangement of its binding partners. Investigating cryo-sections of human skeletal muscle with antibodies against Xin and Xirp2 directly showed for the first time their involvement in skeletal muscle remodelling. Skeletal muscle areas lacking a proper sarcomere arrangement exhibited Xin and Xirp2-containing longitudinal structures connecting adjacent sarcomeres. These strands were not prominently associated with major structural components of the Z-disc such as α-actinin and nebulin while filamin C was newly identified to be associated with these structures. In conclusion, this work provides a detailed view on differences and similarities of Xin-Repeat protein thereby offering further insights into their function. While both Xin and Xirp2 are involved in early events of myogenic differentiation, Xin takes over a rather transient role whereas Xirp2 presumably contributes to a permanent scaffold for Z-disc structure. Their participation in skeletal muscle remodelling render Xin-Repeat proteins important players of myogenic development and remodelling processes

Comparison of magnetic and metabolic brain activity during a verb generation task

The magnetic and metabolic activational patterns of the brain during the perception, generation and silent articulation of words overlap to some extent, yet also measure concrete activational patterns. In the present study, auditory evoked magnetic fields (MEG) and changes in regional cerebral blood low (PET) were examined in healthy subjects during a verb generation task. The aim of the study was to determine whether the advantages of both recording techniques can be combined so as to identify distributed sources of brain activity during particular tasks such as language processing. Given the currently observed disparity of the results from the two types of brain imaging we conclude that PET data will most likely not provide physiologically meaningful constraints for the distributed source analysis of MEG data, and may not necessarily validate results of distributed source analyses

Actin cytoskeleton Xin repeat proteins

available at www.sciencedirect.com www.elsevier.com/locate/yexcr Research Article Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VAS

Recognizing sequences of sequences

The brain's decoding of fast sensory streams is currently impossible to emulate, even approximately, with artificial agents. For example, robust speech recognition is relatively easy for humans but exceptionally difficult for artificial speech recognition systems. In this paper, we propose that recognition can be simplified with an internal model of how sensory input is generated, when formulated in a Bayesian framework. We show that a plausible candidate for an internal or generative model is a hierarchy of 'stable heteroclinic channels'. This model describes continuous dynamics in the environment as a hierarchy of sequences, where slower sequences cause faster sequences. Under this model, online recognition corresponds to the dynamic decoding of causal sequences, giving a representation of the environment with predictive power on several timescales. We illustrate the ensuing decoding or recognition scheme using synthetic sequences of syllables, where syllables are sequences of phonemes and phonemes are sequences of sound-wave modulations. By presenting anomalous stimuli, we find that the resulting recognition dynamics disclose inference at multiple time scales and are reminiscent of neuronal dynamics seen in the real brain