The LIM domain protein nTRIP6 modulates the dynamics of myogenic differentiation

The process of myogenesis which operates during skeletal muscle regeneration involves the activation of muscle stem cells, the so-called satellite cells. These then give rise to proliferating progenitors, the myoblasts which subsequently exit the cell cycle and differentiate into committed precursors, the myocytes. Ultimately, the fusion of myocytes leads to myofiber formation. Here we reveal a role for the transcriptional co-regulator nTRIP6, the nuclear isoform of the LIM-domain protein TRIP6, in the temporal control of myogenesis. In an in vitro model of myogenesis, the expression of nTRIP6 is transiently up-regulated at the transition between proliferation and differentiation, whereas that of the cytosolic isoform TRIP6 is not altered. Selectively blocking nTRIP6 function results in accelerated early differentiation followed by deregulated late differentiation and fusion. Thus, the transient increase in nTRIP6 expression appears to prevent premature differentiation. Accordingly, knocking out the Trip6 gene in satellite cells leads to deregulated skeletal muscle regeneration dynamics in the mouse. Thus, dynamic changes in nTRIP6 expression contributes to the temporal control of myogenesis.

Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin's mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

RhoA mediates stress fiber strain site recognition by the LIM protein testin

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize strained actin while the N-terminal domains alone recognize unstrained actin. Phosphorylation and cancer related mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes WT testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin’s mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state.SummaryLIM domain proteins recognize local strain in the actin cytoskeleton. This work suggests that the conformational state of the LIM protein testin determines its ability to recognize strained stress fibers and reveals a role for RhoA in regulating testin’s mechanosensitivity.

The Cytoplasmic LIM Domain Protein Espinas Contributes to Photoreceptor Layer Selection in the Visual System

During circuit assembly it is essential that neurons connect with their specific synaptic partners. To facilitate this process, a common strategy in many organisms is the organization of brain regions, including the fly visual system, in layers and columns. The atypical-cadherin Flamingo (Fmi) and the receptor Golden Goal (Gogo) were proposed to regulate both the temporary and final layer selection of the R8 photoreceptor, through the cytoplasmic domain of Gogo. Our data suggests that Fmi intracellular signaling is also relevant for R8 final layer selection. The LIM-domain cytoplasmic molecule Espinas (Esn) binds Fmi, and they cooperatively control dendritic self-avoidance in sensory neurons. We observed defects in R8 layer selection in esn mutants with axons overshooting the final target layer, and we demonstrated that the LIM domain is necessary for layer selection. fmi knockdown in photoreceptors results in most R8 axons stalling at the temporary layer, however, we also detected R8 axons projecting past the final-target layer, and showed that fmi and esn genetically interact. Based on the previously described physical and genetic interactions between Fmi/Esn and the findings presented here, we propose that Esn signals downstream of Fmi to stabilize R8 axons in their final target layer.

PDZ and LIM domain protein 2 plays dual and context-dependent roles in breast cancer development

ABSTRACTBackgroundPDZ and LIM domain protein 2 (PDLIM2) is a cytoskeletal and nuclear effector that regulates the activity of several transcription factors (e.g., NF-κB, STAT), and its deregulation has been associated with oncogenesis. Our recent study identified PDLIM2 as a protein associated with the lymph node metastasis of low grade luminal A breast cancer tissues. Here, we aim to understand this association at the molecular and cellular levels.MethodsTo investigate the link between PDLIM2 and epithelial-to-mesenchymal transition (EMT), stably transduced MCF7-PDLIM2 cells, and MCF7 or MCF10A cells with PDLIM2 protein levels modified using siRNA or PDLIM2 gene carrying plasmid, were used. Additionally, MCF7 and MCF10A cells were exposed to hypoxic conditions and TGFβ1 treatment. EMT was monitored using immunoblotting of EMT markers and atomic force microscopy (AFM). The role of PDLIM2 in cell migration and/or invasion was investigated using Transwell assay and xCELLigence system.ResultsFirst, we observe a positive effect of PDLIM2 overexpression on EMT in MCF7 cells, a model of luminal A tumors, using EMT markers and AFM. On the other hand, PDLIM2 helps to maintain the epithelial phenotype in MCF10A cells, a model of normal breast epithelial cells. Second, we find that exposure of the MCF7 cells to hypoxic conditions increases levels of PDLIM2 and carbonic anhydrase-9 (CA-9), a marker of the response to hypoxia. However, none of these effects are observed in the MCF10A cells. Third, PDLIM2 overexpression promotes migration, invasion, and proliferation and decreases adhesion of the MCF7 cells, but an opposite effect is observed in the MCF10A cells.ConclusionsOur data indicate that PDLIM2 plays a dual role: (i) as an EMT-supporting and hypoxia-responding oncoprotein in luminal breast cancer cells, and (ii) as an epithelial phenotype-maintaining tumor suppressor in normal epithelial breast cells.