Timothy Megraw - Academia.edu (original) (raw)

Papers by Timothy Megraw

Journal of Cellular Physiology

Cilia, 2017

La-related protein 6 (LARP6) is an evolutionally conserved RNA-binding protein. Vertebrate LARP6 ... more La-related protein 6 (LARP6) is an evolutionally conserved RNA-binding protein. Vertebrate LARP6 binds the 5' stem-loop found in mRNAs encoding type I collagen to regulate their translation, but other target mRNAs and additional functions for LARP6 are unknown. The aim of this study was to elucidate an additional function of LARP6 and to evaluate the importance of its function during development. To uncover the role of LARP6 in development, we utilized Morpholino Oligos to deplete LARP6 protein in Xenopus embryos. Then, embryonic phenotypes and ciliary structures of LAPR6 morphants were examined. To identify the molecular mechanism underlying ciliogenesis regulated by LARP6, we tested the expression level of cilia-related genes, which play important roles in ciliogenesis, by RT-PCR or whole mount in situ hybridization (WISH). We knocked down LARP6 in Xenopus embryos and found neural tube closure defects. LARP6 mutant, which compromises the collagen synthesis, could rescue these ...

Methods in Molecular Biology, 2016

Hundreds of Drosophila cell lines have been established in the labs of many researchers over the ... more Hundreds of Drosophila cell lines have been established in the labs of many researchers over the last decades and have been important tools for research. Although these cells often deviate from normal cell physiology and genetic composition, such systems nonetheless are powerful models for biochemical, cell biological, and genetics studies that are experimentally difficult in vivo. While published descriptions of cell line generation are available in the literature, how to generate new Drosophila cell lines can be challenging for beginners. Here, we describe a detailed, simple protocol to establish new Drosophila cell lines.

Cilia, 2016

The fruit fly, Drosophila melanogaster, is one of the most extensively studied organisms in biolo... more The fruit fly, Drosophila melanogaster, is one of the most extensively studied organisms in biological research and has centrioles/basal bodies and cilia that can be modelled to investigate their functions in animals generally. Centrioles are nine-fold symmetrical microtubule-based cylindrical structures required to form centrosomes and also to nucleate the formation of cilia and flagella. When they function to template cilia, centrioles transition into basal bodies. The fruit fly has various types of basal bodies and cilia, which are needed for sensory neuron and sperm function. Genetics, cell biology and behaviour studies in the fruit fly have unveiled new basal body components and revealed different modes of assembly and functions of basal bodies that are conserved in many other organisms, including human, green algae and plasmodium. Here we describe the various basal bodies of Drosophila, what is known about their composition, structure and function.

Cells

The centrosome is the best-understood microtubule-organizing center (MTOC) and is essential in pa... more The centrosome is the best-understood microtubule-organizing center (MTOC) and is essential in particular cell types and at specific stages during Drosophila development. The centrosome is not required zygotically for mitosis or to achieve full animal development. Nevertheless, centrosomes are essential maternally during cleavage cycles in the early embryo, for male meiotic divisions, for efficient division of epithelial cells in the imaginal wing disc, and for cilium/flagellum assembly in sensory neurons and spermatozoa. Importantly, asymmetric and polarized division of stem cells is regulated by centrosomes and by the asymmetric regulation of their microtubule (MT) assembly activity. More recently, the components and functions of a variety of non-centrosomal microtubule-organizing centers (ncMTOCs) have begun to be elucidated. Throughout Drosophila development, a wide variety of unique ncMTOCs form in epithelial and non-epithelial cell types at an assortment of subcellular locatio...

Nature communications, Jan 21, 2011

Centrosomes are conserved organelles that are essential for accurate cell division and cilium for... more Centrosomes are conserved organelles that are essential for accurate cell division and cilium formation. A centrosome consists of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM) that is essential for the centrosome's function. In this study, we show that Sas-4 provides a scaffold for cytoplasmic complexes (named S-CAP), which include CNN, Asl and D-PLP, proteins that are all found in the centrosomes at the vicinity of the centriole. When Sas-4 is absent, nascent procentrioles are unstable and lack PCM, and functional centrosomes are not generated. When Sas-4 is mutated, so that it cannot form S-CAP complexes, centrosomes are present but with dramatically reduced levels of PCM. Finally, purified S-CAP complexes or recombinant Sas-4 can bind centrosomes stripped of PCM, whereas recombinant CNN or Asl cannot. In summary, PCM assembly begins in the cytosol where Sas-4 provides a scaffold for pre-assembled cytoplasmic complexes before tethering o...

Advances in skin & wound care, 2016

To examine the efficacy of the folk remedy of chicken-egg membrane dressing on wound healing. Ful... more To examine the efficacy of the folk remedy of chicken-egg membrane dressing on wound healing. Full-thickness excisional wounds were created on 14 male Sprague-Dawley rats in 2 separate trials. Each animal received 2 wounds on the upper back. One wound was untreated, and the other was dressed with chicken-egg membrane to assess its impact on wound healing. Half of the rats received egg membrane treatment on the inferior wound, whereas the other half received egg membrane treatment on the superior wound. Membrane replacement, wound debridement, and imaging were done on days 5, 8, and 10 and then imaging continued on days 12, 14, 16, 18, and 20 of the experiment. Healing rate was measured based on the wound area over the 20 days of the experiment. The wounds dressed with chicken-egg membrane had a significantly (P < .01) faster rate of healing compared with the control at the early stages of healing between days 0 and 5. This group healed 21% faster during this early phase, compared...

European journal of cell biology, Jan 4, 2015

Centrosome cohesion maintains centrosomes in close proximity until mitosis, when cell cycle-depen... more Centrosome cohesion maintains centrosomes in close proximity until mitosis, when cell cycle-dependent regulatory signaling events dissolve cohesion and promote centrosome separation in preparation for bipolar spindle assembly at mitosis. Cohesion is regulated by the antagonistic activities of the mitotic NIMA-related kinase 2 (Nek2), protein phosphatase 1, the cohesion fiber components rootletin, centrosomal Nek2-associated protein 1 (C-Nap1) and Cep68. The centrosomal protein Cep68 is essential for centrosome cohesion and dissociates from centrosomes at the onset of mitosis. Here, our cell line studies show the C-terminal 300-400 amino acids of Cep68 are necessary to localize Cep68 to interphase centrosomes while C-terminal 400-500 amino acids might regulate Cep68 dissociation from centrosomes at mitotic onset. In addition, Nek2 was demonstrated to phosphorylate Cep68 in vivo and this phosphorylation appears to promote Cep68 degradation in mitosis. We further show that the SCF comp...

NeuroMolecular Medicine, 2015

Neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington&#... more Neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases have high prevalence among the elderly. Many strategies have been established to alleviate the symptoms experienced by affected individuals. Recent studies have shown that exercise helps patients with neurological disorders to regain lost physical abilities. PGC1α/FNDC5/BDNF has emerged recently as a critical pathway for neuroprotection. PGC1α is a highly conserved co-activator of transcription factors that preserves and protects neurons against destruction. PGC1α regulates FNDC5 and its processed and secreted peptide Irisin, which has been proposed to play a critical role in energy expenditure and to promote neural differentiation of mouse embryonic stem cells. FNDC5 may also increase the expression of the neurotrophic factor BDNF, a neuroprotective agent, in the hippocampus. BDNF is secreted from hippocampus, amygdala, cerebral cortex and hypothalamus neurons and initiates intracellular signaling pathways through TrkB receptors. These pathways have positive feedback on CREB activities and lead to enhancement in PGC1α expression in neurons. Therefore, FNDC5 could behave as a key regulator in neuronal survival and development. This review presents recent findings on the PGC1α/FNDC5/BDNF pathway and its role in neuroprotection, and discusses the controversial promise of irisin as a mediator of the positive benefits of exercise.

Molecular biology of the cell, Jan 6, 2016

Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600)... more Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here, we characterize the single Nin ortholog inDrosophilaDrosophilaNin localizes to the periphery of the centrosome, but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian ortholog of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to non-centrosomal microtubule-organizing cente...

Gene, 1998

A cDNA synthesized and cloned from Drosophila melanogaster mRNA was shown to encode a protein wit... more A cDNA synthesized and cloned from Drosophila melanogaster mRNA was shown to encode a protein with significant identity to the protein encoded by the Drosophila Antigen 5-related (Agr) gene. Because of its similarity to Agr, this novel cDNA was termed Antigen 5-related 2 (Agr2). The proteins encoded by Agr and Agr2 both belong to the CAP family of proteins, which include the mammalian Cysteine-rich secretory proteins, wasp venom Antigen 5 proteins, and plant group 1 Pathogenesis-related proteins. CAP family proteins share a common core sequence, and most appear to function extracellularly. Agr2 encodes a protein that is 254 amino acids in length. The cDNA contains a short 5′ untranslated region (UTR) 36 bp in length, and a 3′ UTR of 46 bp. The protein encoded by Agr2 has 48% identity and 61% similarity to that encoded by Agr. The Agr2 gene was localized to region 12F of the X chromosome and probably lies near the Agr gene. Agr2 RNA was approx. 950 nt in length and was most abundant during the larval period, although transcripts could also be detected at other developmental stages. Transcription of Agr2 is initiated in the embryo within the midgut epithelium near the site where the third midgut constriction will form. As embryogenesis proceeds, Agr2 expression expands to form a domain located just posterior to the third midgut constriction. This domain corresponds to a single loop of the midgut in late-stage embryos. Based on its expression pattern and its similarity to other CAP family proteins, the Agr2 protein is hypothesized to function either as a novel type of protease inhibitor or as an antimicrobial protein.

Journal of Biological Chemistry, Jun 15, 1993

The mitochondrial histone HM is the major DNAbinding protein in mitochondria and is necessary for... more The mitochondrial histone HM is the major DNAbinding protein in mitochondria and is necessary for maintenance of the mitochondrial genome in the yeast Saccharomyces cerevisiae during growth on fermentable sugars. HM and the Escherichia coli histone-like protein HU have similar activities in vitro, including DNA supercoiling, but share no sequence similarity. We show that HU can functionally complement the respiration deficiency associated with yeast strains lacking HM. Conversely, phenotypes of E. coli cells lacking HU protein, including nucleoid loss and a filamentous cell morphology, were alleviated by expression of HM in these cells. The HU protein of bacteria and the HM protein of mitochondria are therefore functionally complementary in vivo. Functional similarities among HM, HU, and the nuclear HMGl proteins are implicated and discussed.

Mol Biol Cell, 2007

Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microt... more Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microtubule spindle apparatus at mitosis. Although the individual binding steps in centrosome maturation are largely unknown, Centrosomin (Cnn) is an essential mitotic centrosome component required for assembly of all other known pericentriolar matrix (PCM) proteins to achieve microtubule-organizing activity at mitosis in Drosophila. We have identified a conserved motif (Motif 1) near the amino terminus of Cnn that is essential for its function in vivo. Cnn Motif 1 is necessary for proper recruitment of ␥-tubulin, D-TACC (the homolog of vertebrate transforming acidic coiled-coil proteins [TACC]), and Minispindles (Msps) to embryonic centrosomes but is not required for assembly of other centrosome components including Aurora A kinase and CP60. Centrosome separation and centrosomal satellite formation are severely disrupted in Cnn Motif 1 mutant embryos. However, actin organization into pseudocleavage furrows, though aberrant, remains partially intact. These data show that Motif 1 is necessary for some but not all of the activities conferred on centrosome function by intact Cnn.

Methods in Molecular Biology, Feb 1, 2004

The Journal of cell biology, Jan 26, 2015

Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal... more Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal structure called the ciliary rootlet links the cilium to the cell body. Previous studies indicated that rootlets support the long-term stability of some cilia. Here we report that Drosophila melanogaster Rootletin (Root), the sole orthologue of the mammalian paralogs Rootletin and C-Nap1, assembles into rootlets of diverse lengths among sensory neuron subtypes. Root mutant neurons lack rootlets and have dramatically impaired sensory function, resulting in behavior defects associated with mechanosensation and chemosensation. Root is required for cohesion of basal bodies, but the cilium structure appears normal in Root mutant neurons. We show, however, that normal rootlet assembly requires centrioles. The N terminus of Root contains a conserved domain and is essential for Root function in vivo. Ectopically expressed Root resides at the base of mother centrioles in spermatocytes and locali...

Cell Reports, 2015

The cilium is a small cellular organelle with motility- and/or sensory-related functions that pla... more The cilium is a small cellular organelle with motility- and/or sensory-related functions that plays a crucial role during developmental and homeostatic processes. Although many molecules or signal transduction pathways that control cilia assembly have been reported, the mechanisms of ciliary length control have remained enigmatic. Here, we report that Smad2-dependent transforming growth factor β (TGF-β) signaling impacts the length of motile cilia at the Xenopus left-right (LR) organizer, the gastrocoel roof plate (GRP), as well as at the neural tube and the epidermis. Blocking TGF-β signaling resulted in the absence of the transition zone protein B9D1/MSKR-1 from cilia in multi-ciliated cells (MCCs) of the epidermis. Interestingly, this TGF-β activity is not mediated by Mcidas, Foxj1, and RFX2, the known major regulators of ciliogenesis. These data indicate that TGF-β signaling is crucial for the function of the transition zone, which in turn may affect the regulation of cilia length.