Min-Sheng Zhu

Trio Is a Key Guanine Nucleotide Exchange Factor Coordinating Regulation of the Migration and Morphogenesis of Granule Cells in the Developing Cerebellum

Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.

The Transmembrane Protein 16A Ca2+-activated Cl− Channel in Airway Smooth Muscle Contributes to Airway Hyperresponsiveness

RATIONALE Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca(2+)-activated Cl(-) [Cl((Ca))] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown. OBJECTIVES Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl((Ca)) channels, and activation of Cl((Ca)) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl((Ca)) channels in ASM and mediate AHR. METHODS We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl(-) currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca(2+) agonist-induced cell shortening, and on Cl((Ca)) currents activated by Ca(2+) sparks (localized, short-lived Ca(2+) transients due to the opening of ryanodine receptors) in mouse ASM cells. MEASUREMENTS AND MAIN RESULTS TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl((Ca)) currents with kinetics similar to native Cl((Ca)) currents. TMEM16A deletion renders Ca(2+) sparks unable to activate Cl((Ca)) currents, and weakens caffeine- and methacholine-induced cell shortening. CONCLUSIONS Tmem16a encodes Cl((Ca)) channels in ASM and contributes to Ca(2+) agonist-induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.

Activation of BK channels may not be required for bitter tastant-induced bronchodilation

To the Editor: Deshpande et al.1 reported that bitter tastants increase intracellular Ca2+ concentration (to similar levels produced by the bronchoconstrictive agonists histamine and bradykinin) yet cause marked bronchodilation. This implies that elevated Ca2+ concentration inhibits contraction, challenging the classic Ca2+-dependent mechanism underlying smooth muscle contraction2,3. To resolve this apparent paradox, the authors showed that bitter tastants can generate localized Ca2+ events, and that bitter tastantinduced relaxation and hyperpolarization can be inhibited by the largeconductance Ca2+-activated K+ (BK) channel antagonist iberiotoxin1; thus, they propose that bitter tastant–induced bronchodilation results from its ability to generate localized Ca2+ signals, which in turn open BK channels and hyperpolarize the membrane. However, their assertion of the involvement of BK channel activation was solely based on the effect of iberiotoxin on bitter tastant–induced relaxation and change in membrane potential as assessed by voltage-sensitive dyes. We are concerned that the association of BK channel activity with relaxation has not been directly examined, raising questions about the proposed relaxation mechanism. We therefore directly studied the effect of bitter tastants on the activity of BK channels and examined the relaxation effect of multiple BK channel inhibitors in mouse airway smooth muscle, the same type of smooth muscle tissue used in Deshpande et al.1. To directly investigate the effect of bitter tastants on BK channel function, we used patch-clamp technology (see Supplementary Methods), an unequivocal methodology for studying ion channel activity. We first examined the effects of the bitter tastant chloroquine on spontaneous transient outward currents (STOCs). These currents result from the opening of BK channels in response to local, short-lived Ca2+ events, that is, Ca2+ sparks, thus representing reliable readouts of the BK channel activity in these cells4,5. If bitter tastants generate localized Ca2+ events as Deshpande et al.1 have suggested, it is plausible that they would increase the activity of STOCs. However, we found that chloroquine at 1 mM, a dose that causes total relaxation (Fig. 3c in Deshpande et al.1), did not increase STOC amplitude or frequency within the first minute or so of application (Fig. 1a,b; n = 9). To our surprise, about 2 min after application chloroquine began to inhibit and then completely block STOCs (Fig. 1a,b). The inhibition was reversible (Fig. 1a), indicating that chloroquine at this concentration does not cause appreciable damage to the cells. BK channels are gated by both Ca2+ and membrane potential, and hence we investigated the effect of chloroquine on depolarization-evoked K+ currents. Upon depolarization from –70 mV to –15 mV, mouse airway smooth muscle cells produced a peak current of 104 ± 8 pA (Fig. 1c; n = 14). Chloroquine (1 mM) inhibited this current by 35% 2 min after application (Fig. 1c; P < 0.05 with paired t test, n = 8). Similarly, iberiotoxin (100 nM) suppressed this current by 30% (data not shown; P < 0.05 with paired t test, n = 6). These results prompted us to reexamine bitter tastant–induced relaxation and the effect of BK channel blockers on this relaxation in isolated mouse airway. The bitter tastants quinine, chloroquine and denatonium all relaxed methacholine-induced contraction and did so in a concentrationdependent manner (Fig. 2a,b), consistent with the results of Deshpande et al.1. At 1 mM, within 4.5 ± 0.4 min (n = 29), chloroquine relaxed methacholineinduced contraction by 91.3 ± 2.4%. The extent of this relaxation in intrapulmonary mainstem bronchi (Fig. 2b,c) was comparable to that in trachea and extrapulmonary mainstem bronchi (Supplementary Fig. 1a,b). However, chloroquine (1 mM) still fully reversed methacholineinduced contraction in the presence of 100 nM iberiotoxin (Fig. 2d). This is in contrast to the result of Deshpande et al.1, who showed that chloroquine only partially reverses the contraction under the same conditions. The reasons for this discrepancy are not known. However, because of this discrepancy, we also examined the effect of iberiotoxin at 300 nM. We found that at this high concentration iberotoxin still exerted no effect on chloroquine-induced relaxation (Fig. 2d,e). These results indicate that bitter tastants do not activate BK chan100 s Chloro 1 mM Wash

Myosin Light Chain Kinase (MLCK) Regulates Cell Migration in a Myosin Regulatory Light Chain Phosphorylation-independent Mechanism

Background: MLCK in cell migration remains controversial. Results: MLCK deletion causes enhanced cell protrusion along with a reduction of membrane tension and is rescued by kinase-dead MLCK or five-DFRXXL motif. Conclusion: MLCK regulates cell migration not by myosin regulatory light chain phosphorylation but possibly through a membrane tension-based mechanism. Significance: Our results shed light on a novel regulatory mechanism of protrusion during cell migration. Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

Deletion of myosin light chain kinase in endothelial cells has a minor effect on the lipopolysaccharide‐induced increase in microvascular endothelium permeability in mice

There is a current view that myosin light chain kinase (MLCK) plays a critical role in endothelial permeability. To investigate the functions of MLCK in endothelial cells in vivo, we generated a mouse model in which MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2/cre transgenic mice. Knocking out Mylk1 from endothelial cells had no effect on the global phenotype of the mice, including body weight and blood pressure. Lipopolysaccharide (LPS)‐mediated septic death was also not altered in the knockout (KO) mice. Consistently, LPS‐induced inflammatory injury and the increase in microvascular permeability in the main organs, including the lung and the kidney, was not significantly attenuated in KO mice as compared with wild‐type mice. However, the LPS‐induced microvascular hyperpermeability of the esophagus and the eyeballs was attenuated in KO mice. We also found that the LPS‐mediated increase in the number of caveolae in the endothelial cells of the esophagus was significantly reduced in KO mice. Our results do not support a role for endothelial cell MLCK in the pathogenesis of inflammatory diseases.

Outer hair cell–specific prestin-CreERT2 knockin mouse lines

Outer hair cells (OHCs) in the cochlea are crucial for the remarkable hearing sensitivity and frequency tuning. To understand OHC physiology and pathology, it is imperative to use mouse genetic tools to manipulate gene expression specifically in OHCs. Here, we generated two prestin knockin mouse lines: (1) the prestin‐CreERT2 line, with an internal ribosome entry site‐CreERT2‐FRT‐Neo‐FRT cassette inserted into the prestin locus after the stop codon, and (2) the prestin‐CreERT2‐NN line, with the FRT‐Neo‐FRT removed subsequently. We characterized the inducible Cre activity of both lines by crossing them with the reporter lines CAG‐eGFP and Ai6. Cre activity was induced with tamoxifen at various postnatal ages and only detected in OHCs, resembling the endogenous prestin expression pattern. Moreover, prestin‐CreERT2 +/− (heterozygotes) and +/+ (homozygotes) as well as prestin‐CreERT2‐NN +/− mice displayed normal hearing. These two prestin‐CreERT2 mouse lines are therefore useful tools to analyze gene function in OHCs in vivo. genesis 50:124–131, 2012.

The molecular basis of the genesis of basal tone in internal anal sphincter

Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca2+ channels (VDCCs) or TMEM16A Ca2+-activated Cl− channels significantly changes global cytosolic Ca2+ concentration ([Ca2+]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca2+]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca2+]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence.

Regulation of DLK1 by the maternally expressed miR-379/miR-544 cluster may underlie callipyge polar overdominance inheritance

Significance The polar overdominance inheritance of callipyge sheep is an unusual mode of non-Mendelian inheritance. We established a mouse line with deletion of the microRNA (miR) 379/miR-544 cluster and assessed the role of this cluster in the inheritance. Our results showed that the maternally expressed miR-379/miR-544 cluster might regulate skeletal muscle growth through the imprinted Delta-like 1 homolog (Dlk1) gene. This report revealed a molecular mechanism of the polar overdominance inheritance. Inheritance of the callipyge phenotype in sheep is an example of polar overdominance inheritance, an unusual mode of inheritance. To investigate the underlying molecular mechanism, we profiled the expression of the genes located in the Delta-like 1 homolog (Dlk1)–type III iodothyronine deiodinase (Dio3) imprinting region in mice. We found that the transcripts of the microRNA (miR) 379/miR-544 cluster were highly expressed in neonatal muscle and paralleled the expression of the Dlk1. We then determined the in vivo role of the miR-379/miR-544 cluster by establishing a mouse line in which the cluster was ablated. The maternal heterozygotes of young mutant mice displayed a hypertrophic tibialis anterior muscle, extensor digitorum longus muscle, gastrocnemius muscle, and gluteus maximus muscle and elevated expression of the DLK1 protein. Reduced expression of DLK1 was mediated by miR-329, a member of this cluster. Our results suggest that maternal expression of the imprinted miR-379/miR-544 cluster regulates paternal expression of the Dlk1 gene in mice. We therefore propose a miR-based molecular working model for polar overdominance inheritance.

Identification and functional characterization of an aggregation domain in long myosin light chain kinase

The functions of long smooth muscle myosin light chain kinase (L‐MLCK), a molecule with multiple domains, are poorly understood. To examine the existence of further potentially functional domains in this molecule, we analyzed its amino acid sequence with a tango program and found a putative aggregation domain located at the 4Ig domain of the N‐terminal extension. To verify its aggregation capability in vitro, expressible truncated L‐MLCK variants driven by a cytomegalovirus promoter were transfected into cells. As anticipated, only the overexpression of the 4Ig fragment led to particle formation in Colon26 cells. These particles contained 4Ig polymers and actin. Analysis with detergents demonstrated that the particles shared features in common with aggregates. Thus, we conclude that the 4Ig domain has a potent aggregation ability. To further examine this aggregation domain in vivo, eight transgenic mouse lines expressing the 4Ig domain (4Ig lines) were generated. The results showed that the transgenic mice had typical aggregation in the thigh and diaphragm muscles. Histological examination showed that 7.70 ± 1.86% of extensor digitorum longus myofibrils displayed aggregates with a 36.44% reduction in myofibril diameter, whereas 65.13 ± 3.42% of diaphragm myofibrils displayed aggregates and the myofibril diameter was reduced by 43.08%. Electron microscopy examination suggested that the aggregates were deposited at the mitochondria, resulting in structural impairment. As a consequence, the oxygen consumption of mitochondria in the affected muscles was also reduced. Macrophenotypic analysis showed the presence of muscular degeneration characterized by a reduction in force development, faster fatigue, decreased myofibril diameters, and structural alterations. In summary, our study revealed the existence of a novel aggregation domain in L‐MLCK and provided a direct link between L‐MLCK and aggregation. The possible significance and mechanism underlying the aggregation‐based pathological processes mediated by L‐MLCK are also discussed.

A bacterial artificial chromosome transgenic mouse model for visualization of neurite growth.

Class III β-tubulin (Tubb3) is a component of the microtubules in neurons and contributes to microtubule dynamics that are required for axon outgrowth and guidance during neuronal development. We here report a novel bacterial artificial chromosome (BAC) transgenic mouse line that expresses Class III β-tubulin fused to mCherry, an improved monomeric red fluorescent protein, for the visualization of microtubules during neuronal development. A BAC containing Tubb3 gene was modified by insertion of mCherry complementary DNA downstream of Tubb3 coding sequence via homologous recombination. mCherry fusion protein was expressed in the nervous system and testis of the transgenic animal, and the fluorescent signal was observed in the neurons that located in the olfactory bulb, cerebral cortex, hippocampal formation, cerebellum, as well as the retina. Besides, Tubb3-mCherry fusion protein mainly distributed in neurites and colocalized with endogenous Class III β-tubulin. The fusion protein labels Purkinje cell dendrites during cerebellar circuit formation. Therefore, this transgenic line might be a novel tool for scientific community to study neuronal development both in vitro and in vivo.