Biology subjects

Campellone, K.

Publications and source records attributed to Campellone, K..

5 recordsLinked to original sources

bioRxiv2026

Synphilin-1 mitigates autophagy dysfunction, modulates ubiquitinated protein aggregation, and promotes cell survival during proteotoxic stress

The decline of cellular proteostasis is a hallmark of aging and key contributor to neurodegenerative diseases. Protein turnover is controlled by the ubiquitin-proteasome and autophagosome-lysosome systems, but how degradation is coordinated when one of these pathways is compromised is not well understood. To study the regulation of proteostasis, we utilized human fibroblasts with targeted knockouts of the cytoskeletal factors WHAMM and JMY, which control multiple steps in autophagy. We found that cells lacking both WHAMM and JMY accumulated numerous intense foci of ubiquitinated proteins when exposed to proteotoxic stress and relied on proteasomes to clear the foci when the stressor was removed. RNA-seq and immunoblotting revealed that WHAMM/JMY knockout cells increased their expression of Synphilin-1, an -synuclein-interacting protein implicated in Parkinsons Disease. In WHAMM/JMY knockout cells that upregulated endogenous Synphilin-1, and in cell lines engineered to overexpress mCherry-Synphilin-1, ubiquitinated proteins were present in structures containing both Synphilin-1 and proteasomes. RNAi-mediated depletion of Synphilin-1 caused a buildup of ubiquitinated proteins and the ubiquitin-binding adaptor protein SQSTM1/p62, while decreasing cell survival in response to proteotoxic stress. These data suggest that Synphilin-1 plays a pro-survival role in cells with impaired autophagy and functions in the distribution of ubiquitinated cargo during proteasomal degradation.

cell biology↗

bioRxiv2024

Autophagosome turnover requires Arp2/3 complex-mediated maintenance of lysosomal integrity

Autophagy is an intracellular degradation process that maintains homeostasis, responds to stress, and plays key roles in the prevention of aging and disease. Autophagosome biogenesis, vesicle rocketing, and autolysosome tubulation are controlled by multiple actin nucleation factors, but the impact of actin assembly on completion of the autophagic pathway is not well understood. Here we studied autophagosome and lysosome remodeling in fibroblasts harboring an inducible knockout (iKO) of the Arp2/3 complex, an essential actin nucleator. Arp2/3 complex ablation resulted in increased basal levels of autophagy receptors and lipidated membrane proteins from the LC3 and GABARAP families. Under both steady-state and starvation conditions, Arp2/3 iKO cells accumulated abnormally high numbers of autolysosomes, suggesting a defect in autophagic flux. The inability of Arp2/3 complex-deficient cells to complete autolysosome degradation and turnover is explained by the presence of damaged, leaky lysosomes. In cells treated with an acute lysosomal membrane-damaging agent, the Arp2/3-activating protein WHAMM is recruited to lysosomes, where Arp2/3 complex-dependent actin assembly is crucial for restoring intact lysosomal structure. These results establish the Arp2/3 complex as a central player late in the canonical autophagy pathway and reveal a new role for the actin nucleation machinery in maintaining lysosomal integrity.

cell biology↗

bioRxiv2024

WHAMM functions in kidney reabsorption and polymerizes actin to promote autophagosomal membrane closure and cargo sequestration

The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.

cell biology↗

bioRxiv2022

F-actin-rich territories coordinate apoptosome assembly and caspase activation during intrinsic apoptosis

The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death processes is not well understood. Here we show that in response to DNA damage, human cells form juxtanuclear F-actin-rich territories that coordinate the organized progression of apoptosome assembly to caspase activation. These cytoskeletal compartments are created by the actin nucleation factors JMY, WHAMM, and the Arp2/3 complex, and they exclude proteins that inhibit JMY and WHAMM activity. Within the territories, JMY localization overlaps with punctate structures containing the core apoptosome components cytochrome c and Apaf-1. The F-actin-rich areas also encompass initiator caspase-9 and clusters of a cleaved form of executioner caspase-3, while restricting accessibility of the caspase inhibitor XIAP. The clustering and potency of caspase-3 activation are positively regulated by the amount of actin polymerized by JMY and WHAMM. These results indicate that JMY-mediated actin reorganization functions in apoptotic signaling by coupling the biogenesis of apoptosomes to the localized processing of caspases.

cell biology↗

bioRxiv2020

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility

Cell motility is governed by cooperation between the Arp2/3 complex and nucleation factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X-chromosome of multiple animals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-mediated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family proteins. AUTHOR SUMMARYThe actin cytoskeleton is a collection of protein polymers that assemble and disassemble within cells at specific times and locations. Sophisticated cytoskeletal regulators called nucleation factors ensure that actin polymerizes when and where it is needed, and most nucleation factors are members of the Wiskott-Aldrich Syndrome Protein (WASP) family. Several of the 8 known WASP-family proteins function in cell motility, but how the different factors collaborate with one another is not well understood. In this study, we identified WHIMP, a new WASP-family member which is encoded on the X chromosome of a variety of animals. In mouse cells, WHIMP enhances cell motility by assembling actin filaments that push the cell membrane forward. Unexpectedly, WHIMP also activates tyrosine kinase enzymes, proteins that stimulate multiple WASP-family members during motility. Our results open new avenues of research into how nucleation factors cooperate during movement and how the molecular activities that underlie motility differ in distinct cell types and organisms.

cell biology↗