Biology subjects

Megraw, T. L.

Publications and source records attributed to Megraw, T. L..

5 recordsLinked to original sources

bioRxiv2024

Zika virus NS3 drives the assembly of a viroplasm-like structure

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused an epidemic in 2015-2016 in the Americas and raised serious global health concerns due to its association with congenital brain developmental defects in infected pregnancies. Upon infection, ZIKV assembles virus particles in a virus-generated toroidal compartment next to the nucleus called the replication factory, or viroplasm, which forms by remodeling the host cell endoplasmic reticulum (ER). How the viral proteins control viroplasm assembly remains unknown. Here we show that the ZIKV non-structural protein 3 (NS3) is sufficient to drive the assembly of a viroplasm-like structure (VLS) in human cells. NS3 encodes a dual-function protease and RNA helicase. The VLS is similar to the ZIKV viroplasm in its assembly near centrosomes at the nuclear periphery, its deformation of the nuclear membrane, its recruitment of ER, Golgi, and dsRNA, and its association with microtubules at its surface. While sufficient to generate a VLS, NS3 is less efficient in several aspects compared to viroplasm formation upon ZIKV infection. We further show that the helicase domain and not the protease domain is required for optimal VLS assembly and dsRNA recruitment. Overall, this work advances our understanding of the mechanism of viroplasm assembly by ZIKV and likely will extend to other flaviviruses. ImportanceThe Zika virus replicates its genome and assembles virus particles in the cytoplasm within the replication organelle, a large virus-induced compartment also called the viroplasm. It does this in part by remodeling the endoplasmic reticulum. However, how the virus directs the host cell to assemble the viroplasm is mostly unknown. This study shows that Zika virus non-structural protein 3 (NS3) is sufficient to assemble a viroplasm-like structures, and indicates that NS3 has a central role in assembling the viroplasm. Understanding how the virus assembles the viroplasm compartment and NS3s role in it should significantly advance our understanding of the cellular mechanisms of virus infection. This study aims to gain more understanding of the Zika virus and its viroplasm along with the molecular mechanisms for viroplasm assembly which might be shared by other viruses.

cell biology↗

bioRxiv2024

A distinct isoform of Msp300/Nesprin organizes the perinuclear microtubule organizing center in adipose cells

In many cell types, disparate non-centrosomal microtubule-organizing centers (ncMTOCs) replace functional centrosomes and serve the unique needs of the cell types in which they are formed. In Drosophila fat body cells (adipocytes), an ncMTOC is organized on the nuclear surface. This perinuclear ncMTOC is anchored by Msp300, encoded by one of two nesprin-encoding genes in Drosophila. Msp300 and the spectraplakin short stop (shot) are co-dependent for localization to the nuclear envelope to generate the ncMTOC where they recruit the microtubule minus-end stabilizer Patronin (CAMSAP). The Msp300 gene is complex, encoding at least eleven isoforms. Here we show that two Msp300 isoforms, Msp300-PE and -PG, are required and only one, Msp300-PE, appears sufficient for generation of the ncMTOC. Loss of Msp300-PE and -PG results in severe loss of localization of shot and Patronin, disruption of the MT array, nuclear mispositioning and loss of endosomal trafficking. Furthermore, upon loss of Msp300-PE and -PG, other isoforms are retained at the nuclear surface despite the loss of nuclear positioning and MT organization, indicating that they are not sufficient to generate the ncMTOC. Msp300-PE has an unusual domain structure including a lack of a KASH domain and very few spectrin repeats and appears therefore to have a highly derived function to generate an ncMTOC on the nuclear surface.

cell biology↗

bioRxiv2023

Ninein domains required for its localization, association with partners dynein and ensconsin, and microtubule organization

Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize microtubules at non-centrosomal microtubule-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here we dissect the protein domains involved in Nins localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates microtubule assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nins association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.

cell biology↗

bioRxiv2022

Evidence for intraflagellar transport in butterfly spermatocyte cilia

In the model organism insect Drosophila melanogaster short cilia assemble on spermatocytes that elaborate into 1.8 mm long flagella during spermatid differentiation. A unique feature of these cilia/flagella is their lack of dependence on intraflagellar transport (IFT) for their assembly. Here we show that in the common butterfly Pieris brassicae the spermatocyte cilia are exceptionally long: about 40 {micro}m compared to less than 1 {micro}m in Drosophila. By transmission electron microscopy we show that P. brassicae spermatocytes display several features not found in melanogaster, including compelling evidence of IFT structures and features of motile cilia. Summary statementThis work shows ultrastructural definition of the exceptionally long cilia that persist on butterfly (P. brassicae) spermatocytes, including evidence of intraflagellar transport, during meiotic division.

cell biology↗

bioRxiv2019

A perinuclear microtubule-organizing center controls nuclear positioning and basement membrane secretion

Non-centrosomal microtubule-organizing centers (ncMTOCs) have a variety of roles presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization, and function. Here we report a novel perinuclear MTOC in Drosophila fat body cells that is anchored by Msp300/Nesprin at the cytoplasmic surface of the nucleus. Msp300 recruits the MT minus-end protein Patronin/CAMSAP, which functions redundantly with Ninein to further recruit the MT polymerase Msps/XMAP215 to assemble non-centrosomal MTs and does so independently of the widespread MT nucleation factor{gamma} -tubulin. Functionally, the fat body ncMTOC and the radial MT arrays it organizes is essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture and MT regulation properties that serves vital functions. HighlightsO_LIA novel perinuclear MTOC in differentiated fat body cells C_LIO_LIThe predominant nucleator,{gamma} -tubulin, is not required at the fat body ncMTOC C_LIO_LIMsp300/Nesprin organizes the ncMTOC at the nuclear surface by recruiting Patronin/CAMSAP and the spectraplakin Shot C_LIO_LIPatronin cooperates with Ninein to control MT assembly at the fat body ncMTOC by recruiting Msps C_LIO_LIMsps, a MT polymerase, is essential for radial MT elongation from the fat body ncMTOC C_LIO_LIPatronin and Msps associate C_LIO_LIThe ncMTOC and radial MTs, but not actin, control nuclear positioning in the fat body C_LIO_LIThe fat body MTOC controls retrograde endocytic trafficking to regulate plasma membrane growth and secretion of basement membrane proteins C_LI O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/888065v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@19822feorg.highwire.dtl.DTLVardef@1216914org.highwire.dtl.DTLVardef@79d0d7org.highwire.dtl.DTLVardef@17dd8a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗