Biology subjects

Penfield, L.

Publications and source records attributed to Penfield, L..

5 recordsLinked to original sources

bioRxiv2025

Hyperactive Rac converts sublethal nibbling to lethal phagocytosis in vivo

The small GTPase Rac is an essential regulator of cell shape, migration, macropinocytosis and phagocytosis. We recently reported that expression of constitutively active RacG12V is sufficient to cause a few migratory cells called border cells to cannibalize nurse cells in the Drosophila ovary. Building on that insight, we engineered mammalian Rac-enhanced chimeric-antigen-receptor macrophages (RaceCAR-Ms) to avidly engulf and kill cancer cells. Here we investigate the cellular and molecular mechanisms by which border cells efficiently kill the much larger nurse cells. Surprisingly, wild type border cells normally nibble on nurse cells as they migrate between them, and RacG12V causes border cells to take larger, lethal bites. These larger bites trigger rapid germline shrinkage, nuclear damage, and caspase activation, which spreads through the nurse cell syncytium. Then, many somatic follicle cells join in to engulf the dying germline. Rac and the engulfment receptor Draper are critical for both sublethal nibbling and lethal phagocytosis. Using clonal analysis, we show small groups of follicle cells expressing RacG12V induced caspase activation in neighboring follicle cells while larger RacG12V clones were required to cause germline killing. Increasing Draper expression or JNK activity in border cells also caused germline death, in a Rac-independent manner, suggesting that border cells can be activated to kill through multiple mechanisms. The series of events elucidated here reveals how hyperactivated Rac expressed in a few cells can trigger destruction of a much larger mass of cells. Significance StatementRac is a key protein in the cellular eating process called phagocytosis. Rac hyperactivity enhances the consumption of tumor cells by chimeric antigen receptor-macrophages (CAR-M), a promising type of cellular immunotherapy. Elucidating the mechanisms by which hyperactive Rac enhances cell killing may lead to improvements in CAR-M. Key insights into the in vivo effects of Rac have come from studying Drosophila oogenesis. Here we report molecular and cellular mechanisms by which hyperactivated Rac stimulates migratory cells to engulf and kill much larger cells in the fly ovary, ultimately resulting in destruction of the entire tissue. These insights have implications for how hyperactivating Rac might improve antibody and CAR-M therapies for cancer and other diseases.

cell biology↗

bioRxiv2023

Nuclear envelope assembly relies on CHMP-7 in the absence of BAF-LEM-mediated hole closure

Barrier-to-autointegration factor (BAF) is a DNA binding protein that crosslinks chromatin to assemble the nuclear envelope (NE) after mitosis. BAF also binds the Lap2b-Emerin-Man1 (LEM) domain family of NE proteins to repair interphase ruptures. The NE adaptors to ESCRTs, LEMD2-CHMP7, seal NE holes surrounding mitotic spindle microtubules (MTs), but whether NE hole closure in mitosis involves BAF-LEM binding is not known. Here, we analyze NE sealing after meiosis II in C. elegans oocytes to show that BAF-LEM binding and LEM-2LEMD2-CHMP-7 have distinct roles in hole closure around spindle MTs. LEM-2/EMR-1emerin function redundantly with BAF-1 to seal the NE. Compromising BAF-LEM binding revealed an additional role for EMR-1 in maintenance of the NE permeability barrier and an essential role for LEM-2-CHMP-7 in preventing NE assembly failure. The WH domain of LEM-2 recruits the majority of CHMP-7 to the NE in C. elegans and a LEM-2 -independent pool of CHMP-7, which is mostly enriched in the nucleoplasm, also contributes to NE stability. Thus, NE hole closure surrounding spindle MTs requires redundant mechanisms that safeguard against failure in NE assembly to support embryogenesis.

cell biology↗

bioRxiv2021

Nuclear lamins promote protrusion dynamics and collective, confined migration in vivo

Cells migrate collectively through confined environments during development and cancer metastasis. The nucleus, a stiff organelle, impedes single cells from squeezing into narrow channels within artificial environments, but how nuclei affect collective cell migration into compact tissues in vivo is unknown. Here, we use border cells in the fly ovary to study nuclear dynamics in collective, confined in vivo migration. Border cells delaminate from the follicular epithelium and squeeze into tiny spaces between cells called nurse cells. The lead cell nucleus transiently deforms as it extends into the lead cell protrusion, which then widens. The nuclei of follower cells deform less. Depletion of the Drosophila B-type lamin, Lam, compromises nuclear integrity, hinders expansion of a leading protrusion, and impedes border cell movement. Cortical myosin II accumulates behind the nucleus and moves it into the protrusion in wildtype but not Lam-depleted cells. These data suggest that the nucleus stabilizes lead cell protrusions to help cells wedge open spaces between nurse cells. SummaryDuring Drosophila border cell migration, the B-type lamin Lam maintains nuclear envelope integrity, stabilizes the lead cell protrusion, and promotes cluster invasion between nurse cells. The nucleus may function as a wedge to prom collective, confined in vivo movement.

cell biology↗

bioRxiv2019

Local regulation of lipid synthesis controls ER sheet insertion into nuclear envelope holes to complete nuclear closure

The nuclear permeability barrier depends on closure of holes in the nuclear envelope (NE). Here, we use meiotic C. elegans oocytes to demonstrate that local control of glycerophospholipid synthesis by CNEP-1/CTDNEP1 regulates the insertion of ER sheets into NE holes and functions independently of ESCRT-III to ensure NE closure. Deletion of CNEP-1 causes excess incorporation of ER membranes into NE holes and a defective NE permeability barrier. ESCRT-III components accumulate at the NE opening surrounding the meiotic spindle, and loss of NE adaptors for ESCRT-III exacerbates NE sealing defects in cnep-1 mutants. Limiting ER sheet production by restoring glycerophospholipid synthesis in cnep-1 mutants rescued NE permeability defects. 3D analysis showed that membrane sheets feed into and narrow NE holes occluded by meiotic spindle microtubules supporting a role for ER sheet insertion in NE closure. Thus, feeding of ER sheets into NE holes must be coordinated with production of ER sheets near the NE to promote NE closure.

cell biology↗

bioRxiv2017

Dynein Pulling Forces On Ruptured Nuclei Counteract Lamin-Mediated Nuclear Envelope Repair Mechanisms In Vivo

Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) undergoes ruptures leading to transient mixing of nuclear and cytoplasmic components. The duration of transient NE ruptures depends on lamins, however the underlying mechanisms and the relevance to in vivo events is not known. Here, we use C. elegans embryos to show that dynein forces that position nuclei increase the severity of lamin-induced NE ruptures in vivo. In the absence of dynein forces, lamin prevents nuclear-cytoplasmic mixing caused by NE ruptures. By monitoring the dynamics of NE rupture events, we demonstrate that lamin is required for a distinct step in NE recovery that restricts nucleocytoplasmic mixing prior to the full restoration of NE rupture sites. We show that laser-induced puncture of the NE recapitulates phenotypes associated with NE recovery in wild type cells. Surprisingly, we find that embryonic lethality does not correlate with the incidence of NE rupture events suggesting that embryos survive transient losses of NE compartmentalization during early embryogenesis. In addition to presenting the first mechanistic analysis of transient NE ruptures in vivo, this work demonstrates that lamin controls the duration of NE ruptures by opposing dynein forces on ruptured nuclei to allow reestablishment of the NE permeability barrier and subsequent restoration of NE rupture sites.

developmental biology↗