Research Symposium

BIO

Jenna is a third year student on a pre-med track.

A KASH-less Msp300 isoform has a novel outer nuclear membrane targeting domain and scaffolds the non-centrosomal microtubule organizing center in fat body cells.

Abstract

In Drosophila fat body cells, nucleus-associated non-centrosomal microtubule-organizing centers (ncMTOCs) coordinate nuclear positioning and intracellular trafficking. The nesprin homolog Msp300 anchors this perinuclear ncMTOC and recruits the microtubule minus-end stabilizer Patronin and other microtubule regulators. Among the 11 Msp300 isoforms, we show that isoform Msp300-PE is necessary and sufficient for ncMTOC formation, despite lacking a canonical KASH domain that anchors conventional nesprins to the outer nuclear membrane (ONM) through association with a SUN domain protein anchored in the inner nuclear membrane (INM). To dissect its unique function in organizing the ncMTOC, we are investigating Msp300-PE localization in fat body cells and examining the phenotypic consequences of targeted CRISPR truncation mutants, focusing on its role in microtubule organization and nuclear positioning. We demonstrate that the unique C-terminus of Msp300-PE, comprising a putative transmembrane helix followed by a 35-aa domain that resides within the nuclear intermembrane space, is sufficient to target the ONM. This finding identifies a novel ONM-targeting domain at the Msp300-PE C-terminus with no sequence similarity to the canonical KASH domain. Notably, this short tail contains seven Cys residues that we propose form disulfide bridges with an unknown partner residing at the INM, a hypothesis supported by non-reducing SDS-PAGE. We are employing mass spectrometry and genetic screens to identify the putative INM partner required for Msp300-PE anchoring and ncMTOC function. This study aims to define the molecular mechanisms by which Msp300-PE, together with its unique domains and partners, generates a perinuclear ncMTOC in fat body cells.

Keywords: KASH, Drosophila, LINC, nuclear, membrane, partner