Comprehensive phenotypic characterization of an allelic series of zebrafish models of NEB-related nemaline myopathy.

Nemaline myopathy (NM) is a rare congenital neuromuscular disorder characterized by muscle weakness and hypotonia, slow gross motor development, and decreased respiratory function. Mutations in at least twelve genes, all of each encode proteins that are either components of the muscle thin filament or regulate its length and stability, have been associated with NM. Mutations in Nebulin (NEB), a giant filamentous protein localized in the sarcomere, account for more than 50% of NM cases.

Lysosomes and the pathogenesis of merosin-deficient congenital muscular dystrophy.

Congenital muscular dystrophy type 1A (MDC1A), the most common congenital muscular dystrophy in Western countries, is caused by recessive mutations in LAMA2, the gene encoding laminin alpha 2. Currently, no cure or disease modifying therapy has been successfully developed for MDC1A. Examination of patient muscle biopsies revealed altered distribution of lysosomes.

The NEMP family supports metazoan fertility and nuclear envelope stiffness.

Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in () with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in , , zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line.

De novo phosphoinositide synthesis in zebrafish is required for triad formation but not essential for myogenesis.

Phosphoinositides (PIPs) and their regulatory enzymes are key players in many cellular processes and are required for aspects of vertebrate development. Dysregulated PIP metabolism has been implicated in several human diseases, including a subset of skeletal myopathies that feature structural defects in the triad. The role of PIPs in skeletal muscle formation, and particularly triad biogenesis, has yet to be determined.

Zebrafish Models of -Related Congenital Muscular Dystrophy (MDC1A).

LAMA2-related congenital muscular dystrophy (CMD; LAMA2-MD), also referred to as merosin deficient CMD (MDC1A), is a severe neonatal onset muscle disease caused by recessive mutations in the LAMA2 gene. LAMA2 encodes laminin α2, a subunit of the extracellular matrix (ECM) oligomer laminin 211. There are currently no treatments for MDC1A, and there is an incomplete understanding of disease pathogenesis.

Insights into wild-type dynamin 2 and the consequences of DNM2 mutations from transgenic zebrafish.

Dynamin 2 (DNM2) encodes a ubiquitously expressed large GTPase with membrane fission capabilities that participates in the endocytosis of clathrin-coated vesicles. Heterozygous mutations in DNM2 are associated with two distinct neuromuscular disorders, Charcot-Marie-Tooth disease (CMT) and autosomal dominant centronuclear myopathy (CNM). Despite extensive investigations in cell culture, the role of dynamin 2 in normal muscle development is poorly understood and the consequences of DNM2 mutations at the molecular level in vivo are not known.

Phosphatidylinositol 4,5-bisphosphate regulates cilium transition zone maturation in .

Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood.

Phosphoinositide signaling in sperm development.

Phosphatidylinositol phosphates (PIPs) are membrane lipids with crucial roles during cell morphogenesis, including the establishment of cytoskeletal organization, membrane trafficking, cell polarity, cell-cycle control and signaling. Recent studies in mice (Mus musculus), fruit flies (Drosophila melanogaster) and other organisms have defined germ cell intrinsic requirements for these lipids and their regulatory enzymes in multiple aspects of sperm development. In particular, PIP levels are crucial in germline stem cell maintenance, spermatogonial proliferation and survival, spermatocyte cytokinesis, spermatid polarization, sperm tail formation, nuclear shaping, and production of mature, motile sperm.

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively.

Adaptive regulation of testis gene expression and control of male fertility by the Drosophila hairpin RNA pathway. [Corrected].

Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs.

The atypical cadherin fat directly regulates mitochondrial function and metabolic state.

Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation.

Drosophila spermiogenesis: Big things come from little packages.

Drosophila melanogaster spermatids undergo dramatic morphological changes as they differentiate from small round cells approximately 12 μm in diameter into highly polarized, 1.8 mm long, motile sperm capable of participating in fertilization. During spermiogenesis, syncytial cysts of 64 haploid spermatids undergo synchronous differentiation.

Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis.

Clathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm.

Phosphatidylinositol 4,5-bisphosphate directs spermatid cell polarity and exocyst localization in Drosophila.

During spermiogenesis, Drosophila melanogaster spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. Here we identify phosphoinositides as critical regulators of these processes.

Depletion of plasma membrane PtdIns(4,5)P2 reveals essential roles for phosphoinositides in flagellar biogenesis.

Axonemes are microtubule-based organelles of crucial importance in the structure and function of eukaryotic cilia and flagella. Despite great progress in understanding how axonemes are assembled, the signals that initiate axoneme outgrowth remain unknown. Here, we identified phosphatidylinositol phosphates (phosphoinositides) as key regulators of early stages of axoneme outgrowth in Drosophila melanogaster spermatogenesis.

Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes.

We tested whether the mechanisms of chromosome movement during anaphase in locust (Locusta migratoria L.) spermatocytes might be similar to those described for crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes, as indicated by the effects of inhibitors and by the localisations of actin and myosin in spindles.

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator.

Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa).

Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis.

Background: Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER.

Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement.

Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator.

Redundant mechanisms for anaphase chromosome movements: crane-fly spermatocyte spindles normally use actin filaments but also can function without them.

Actin inhibitors block or slow anaphase chromosome movements in crane-fly spermatocytes, but stopping of movement is only temporary; we assumed that cells adapt to loss of actin by switching to mechanism(s) involving only microtubules. To test this, we produced actin-filament-free spindles: we added latrunculin B during prometaphase, 9-80 min before anaphase, after which chromosomes generally moved normally during anaphase. We confirmed the absence of actin filaments by staining with fluorescent phalloidin and by showing that cytochalasin D had no effect on chromosome movement.

Does 2,3-butanedione monoxime inhibit nonmuscle myosin?

BDM (2,3-butanedione monoxime) has been used extensively to inhibit nonmuscle myosin. However, recent articles raise the question of what BDM actually does, because of experiments in which BDM did not affect the actin-activated ATPase of nonmuscle myosins. We describe results that indicate that BDM indeed inhibits motility due to nonmuscle myosins: in many different cells BDM has the same effects as anti-actin agents and/or as other anti-myosin agents, and BDM slows or stops the sliding between actin filaments and myosin in vitro.

Continuous phosphatidylinositol metabolism is required for cleavage of crane fly spermatocytes.

Successful cleavage of animal cells requires co-ordinated regulation of the actomyosin contractile ring and cleavage furrow ingression. Data from a variety of systems implicate phosphoinositol lipids and calcium release as potential regulators of this fundamental process. Here we examine the requirement for various steps of the phosphatidylinositol (PtdIns) cycle in dividing crane fly (Nephrotoma suturalis) spermatocytes.