Biology subjects

Hardin, K. R.

Publications and source records attributed to Hardin, K. R..

4 recordsLinked to original sources

bioRxiv2025

A Critical Role for the Fascin Family of Actin Bundling Proteins in Axon Development, Brain Wiring and Function

Actin-based cell motility drives many neurodevelopmental events including guided axonal growth. Fascin is a major family of F-actin bundling proteins, but its role in axon development in vivo and brain wiring remains unclear. Here, we report that fascin is required for axon development, brain wiring and function. We show that fascin is enriched in the motile filopodia of axonal growth cones and its inhibition impairs axonal extension and branching of hippocampal neurons in culture. We next provide evidence that fascin is essential for axon development and brain wiring in vivo using Drosophila melanogaster as a model. Drosophila expresses a single ortholog of mammalian fascin called Singed (SN), which is expressed in the mushroom body (MB) of the central nervous system. Loss of SN causes severe MB disruption, marked by - and {beta}-lobe defects indicative of altered axonal guidance. SN-null flies also exhibit defective sensorimotor behaviors as assessed by the negative geotaxis assay. MB-specific expression of SN in SN-null flies rescues MB structure and sensorimotor deficits, indicating that SN functions autonomously in MB neurons. Together, our data from primary neuronal culture and in vivo models highlight a critical role for fascin in brain development and function. HighlightsO_LIFascin regulates axon growth and branching of hippocampal neurons in culture. C_LIO_LISinged, a Drosophila fascin ortholog, is enriched in mushroom body (MB) axons. C_LIO_LISinged loss causes axon guidance defects and sensorimotor issues in flies. C_LIO_LIMB-specific Singed re-expression rescues MB structure and behavior in flies. C_LI

cell biology↗

bioRxiv2021

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E

The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogs in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in MEFs results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 KO in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other IFT proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi-cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi. SummaryPhylogenetic analyses of ARF family GTPases predict that ARL16 is linked to cilia. This was confirmed using MEFs deleted for ARL16, resulting in defects in Golgi to cilium traffic, with accumulation of IFT140 and INPP5E at Golgi.

cell biology↗

bioRxiv2021

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and Cilia to Regulate Ciliogenesis and Ciliary Protein Traffic

ELMODs are a family of three mammalian paralogs that display GTPase activating protein (GAP) activity towards a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogs ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon expression of activating mutants of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions. Thus, we believe that ELMOD1 and ELMOD3 perform multiple functions in cells, most prominently linked to ciliary biology and Golgi-ciliary traffic, and likely acting from more than one cellular location.

cell biology↗

bioRxiv2021

Mitochondrial energy dysfunction induces remodeling of the cardiac mitochondrial protein acylome

Mitochondria are increasingly recognized as signaling organelles because, under conditions of stress, mitochondria can trigger various signaling pathways to coordinate the cells response. The specific pathway(s) engaged by mitochondria in response to defects in mitochondrial energy production in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. In heart tissue from these mice, mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased post-translational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel crosstalk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the mitochondrial energy production machinery can have an expanded impact on global mitochondrial function.

cell biology↗