Biology subjects

Higgs, H.

Publications and source records attributed to Higgs, H..

8 recordsLinked to original sources

bioRxiv2026

A minimally modified AAV9 variant (AAV9fyhr) for enhanced CNS gene delivery by intra-CSF administration.

Adeno-associated virus (AAV) mediated gene therapies continue to show promise for the treatment of genetic diseases. However, delivery and safety still represent significant prohibitive barriers restricting translation of these delivery modalities to humans. We describe a novel AAV9 variant, AAV9fyhr or FYHR, identified from screening a controlled randomization point mutation AAV9 library in mice and nonhuman primates followed by directed evolution of in vivo CNS-derived libraries screened in human iPSC differentiated cortical neurons. Capsid variants of interest were identified by coupling long read sequencing and computational structure modeling. Structure-function analysis of our lead candidate reveal that two targeted amino acid changes are sufficient to result in beneficial pleiotropic effects across viral stability, cell surface attachment, and gene delivery. We developed a novel multiplexed barcoding reporter system, enabling a time and cost-effective option to gather spatially resolved single cell capsid validation data in mice and nonhuman primates. When administered intrathecally, AAV9fyhr exhibits 2-to-6-fold higher gene delivery across several tissues in the CNS while minimally altering the tropism to peripheral tissues in comparison to AAV9. These findings demonstrate the potential of whole capsid engineering for the rational development of translational next generation AAV capsids.

bioengineering↗

bioRxiv2025

Mobility-enhanced virus vectors enable meristem genome editing in model and crop plants

CRISPR/Cas9 gene editing revolutionized genetics, but its application is often hampered in non-model plants that are recalcitrant or less amenable to standard plant transformation and regeneration methods. Harnessing viruses to convey guide RNAs (gRNAs) directly to the meristem promises to overcome those limitations and accelerate the generation of edited lines in diverse crops. With several RNA viruses, delivery of gRNAs to the meristem is enhanced with the addition of mobile RNA elements. We hypothesized that incorporating distinct RNA secondary structures as candidate mobility factors in the widely used Tobacco rattle virus (TRV) could propel virus delivery for enhanced meristem editing in non-model species. To test this, we engineered TRV vectors to deliver gRNAs targeting visible marker genes, with each virus incorporating unique mobility factors. We determined optimal virus construction for multiplexed meristem editing by first delivering each virus to Nicotiana benthamiana plants harboring the Cas9 transgene. Strikingly different phenotypes were observed among virus treatments, which were confirmed to represent distinct somatic and heritable editing events. We further tested our hypothesis by leveraging these results to edit pennycress (Thlaspi arvense), an emerging oilseed crop. Our results demonstrated successful virus delivery of meristem editing to this non-model plant, underscoring the potential of this approach to deliver targeted genome modifications in diverse crops. One sentence summaryIncorporating RNA mobility factors in TRV affects meristem editing in model plants and crops.

plant biology↗

bioRxiv2025

The tumour suppressor LACTB remodels mitochondria to promotecytochrome c release and apoptosis

Mitochondria are pivotal regulators of cellular homeostasis, integrating energy metabolism, biosynthesis, and programmed cell death (apoptosis). During apoptosis, mitochondrial outer membrane permeabilization by BAX/BAK pores facilitates release of apoptotic factors, while the role of inner mitochondrial membrane (IMM) remodelling remains less understood. Here, we identify LACTB, a filament-forming serine protease and tumour suppressor, as a regulator of IMM dynamics during apoptosis. LACTB is required for apoptosis-induced IMM remodelling, which in turn causes increased release of cytochrome c and other mitochondrial apoptotic factors. LACTB-induced membrane remodelling is independent of OPA1 processing. Rather, purified LACTB binds and remodels cardiolipin-enriched membrane nanotubes preferentially over planar lipid membranes, suggesting a direct effect in apoptotic membrane remodelling. Intriguingly, LACTB is not required for mitochondrial shape changes induced by mitochondrial depolarization, suggesting that LACTB action is apoptosis-specific. Collectively, our findings establish LACTB as a mediator of apoptosis-induced IMM remodelling, suggesting a mechanism for tumour suppression in cancer.

cell biology↗

bioRxiv2023

Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin

Drp1 is a dynamin family GTPase that is required for mitochondrial and peroxisomal division, in which it oligomerizes into a ring and constricts the underlying membrane in a GTP hydrolysis-dependent manner. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600. Phosphorylation of S579 is widely regarded as activating, while S600 phosphorylation is commonly considered inhibiting. However, the direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. In this study, we compare the effects of S579 and S600 phosphorylation on purified Drp1, using phospho-mimetic mutants and in vitro phosphorylation. The oligomerization state of both phospho-mimetic mutants is shifted toward smaller oligomers. Both phospho-mimetic mutations maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. Phosphorylation of S579 by Erk2 produces similar effects. When mixed with wild-type Drp1, both S579D and S600D phospho-mimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant that lacks GTPase activity, the K38A mutant, stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions, similar to previous results for dynamin-1. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly, and likely requires additional factors for stimulation of mitochondrial fission in cells. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.

biochemistry↗

bioRxiv2022

Mitochondrial dysfunction triggers actin polymerization necessary for rapid glycolytic activation

Mitochondrial damage represents a dramatic change in cellular homeostasis, necessitating rapid responses. One rapid response is peri-mitochondrial actin polymerization, termed ADA (acute damage-induced actin). The consequences of ADA are not fully understood. Here we show that ADA is necessary for rapid glycolytic activation upon inhibition of mitochondrial ATP production in multiple cells, including mouse embryonic fibroblasts and effector CD8+ T lymphocytes, for which glycolysis is an important source of ATP and biosynthetic molecules. Treatments that induce ADA include CCCP, antimycin A, rotenone, oligomycin, and hypoxia. The Arp2/3 complex inhibitor CK666 or the mitochondrial sodium-calcium exchanger (NCLX) inhibitor CGP37157, applied simultaneously with the ADA stimulus, inhibit both ADA and the glycolytic increase within 5-min, suggesting that ADA is necessary for glycolytic stimulation. Two situations causing chronic reductions in mitochondrial ATP production, ethidium bromide treatment (to deplete mitochondrial DNA) and mutation to the NDUFS4 subunit of complex 1 of the electron transport chain, cause persistent peri-mitochondrial actin filaments of similar morphology to ADA. Both peri-mitochondrial actin loss and a 20% ATP decrease occur within 10 min of CK666 treatment in NDUFS4 knock-out cells. We propose that ADA is necessary for rapid glycolytic activation upon mitochondrial impairment, to re-establish ATP production.

cell biology↗

bioRxiv2019

Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

Recent studies show that mitochondria and actin filaments work together in two contexts: 1) increased cytoplasmic calcium induces cytoplasmic actin polymerization that stimulates mitochondrial fission, and 2) mitochondrial depolarization causes actin assembly around mitochondria, with roles in mitophagy. It is unclear whether these two processes utilize similar actin assembly mechanisms. Here, we show that these are distinct actin assembly mechanisms in the acute phase after treatment (<10 min). Calcium-induced actin assembly is INF2-dependent and Arp2/3 complex-independent, whereas depolarization-induced actin assembly is Arp2/3 complex-dependent and INF2-independent. The two types of actin polymerization are morphologically distinct, with calcium-induced filaments throughout the cytosol and depolarization-induced filaments as \"clouds\" around depolarized mitochondria. We have previously shown that calcium-induced actin stimulates increases in both mitochondrial calcium and recruitment of the dynamin GTPase Drp1. In contrast, depolarization-induced actin is temporally-associated with extensive mitochondrial dynamics that do not result in mitochondrial fission, but in circularization of the inner mitochondrial membrane (IMM). These dynamics are dependent upon the protease Oma1 and independent of Drp1. Actin cloud inhibition causes increased IMM circularization, suggesting that actin clouds limit these dynamics.\n\nSummary statementMitochondrial depolarization induces Arp2/3 complex-dependent actin clouds that restrain mitochondrial shape changes induced by Oma1 on the inner mitochondrial membrane. A distinct actin network stimulates mitochondrial fission in response to calcium.

cell biology↗

bioRxiv2019

Proteolytic program-dependent functions are impaired in INF2-mediated focal segmental glomerulosclerosis.

Regulation of the actin cytoskeleton is critical for normal glomerular podocyte structure and function. Altered regulation of the podocyte cytoskeleton can lead to proteinuria, reduced kidney filtration function and focal segmental glomerulosclerosis (FSGS). Mutations in inverted formin 2 (INF2), a member of the formin family of actin regulatory proteins, are the most common cause of autosomal dominant FSGS. INF2 is a multi-domain protein regulated by interaction between its N-terminal Diaphanous Inhibitory Domain (DID) and its C-terminal Diaphanous Auto-regulatory Domain (DAD). Although many aspects of the INF2 DID-DAD interaction are understood, it remains unclear why disease-causing mutations are restricted to the DID and how these mutations cause human disease. Here we report a proteolytic cleavage in INF2 that liberates the INF2 N-terminal DID to function independently of the INF2 C-terminal fragment containing the DAD domain. N-terminal DID region epitopes are differentially localized to podocyte foot process structures in normal glomeruli. This N-terminal fragment localization is lost in INF2-mediated FSGS, whereas INF2 C-terminal fragment epitopes localize to the podocyte cell body in both normal and disease conditions. INF2 cleavage is mediated by cathepsin proteases. In cultured podocytes, the wild-type INF2 N-terminal fragment localizes to membrane regions and promotes cell spreading, while these functions are impaired in a disease-associated INF2 mutant R218Q in the DID. These features are dependent on INF2-cleavage, with accompanying interaction of INF2 N-fragment with mDIA1. Our data suggest a unique cellular function of the DID dependent on INF2 cleavage and help explain the altered localization of FSGS-associated INF2 mutant polypeptides.

pathology↗

bioRxiv2018

A complex containing lysine-acetylated actin inhibits the formin INF2

INF2 is a member of the formin family of actin assembly factors. Dominant mis-sense mutations in INF2 link to two diseases: focal segmental glomerulosclerosis (FSGS), a kidney disease; and Charcot-Marie-Tooth disease (CMTD), a neuropathy. All disease mutations map to the autoinhibitory Diaphanous Inhibitory Domain (DID). Curiously, purified INF2 is not autoinhibited, suggesting the existence of additional cellular inhibitors. We purified an INF2 inhibitor from mouse brain, and identified it as a complex between lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP/KAc-actin requires INF2 DID. Treatment of CAP/KAc-actin with histone deacetylase 6 (HDAC6) releases INF2 inhibition, while HDAC6 inhibitors block cellular INF2 activation. INF2 disease mutants are poorly inhibited by CAP/KAc-actin, suggesting that FSGS and CMTD result from reduced CAP/KAc-actin binding. This is the first demonstrated role for lysine-acetylated actin: regulation of an actin assembly factor by a novel mechanism, which we call facilitated auto-inhibition.

biochemistry↗