Biology subjects

Anton, Z.

Publications and source records attributed to Anton, Z..

5 recordsLinked to original sources

bioRxiv2026

Kinase-gated coincidence detection controls kinesin-driven lysosome transport

Kinesin motors drive long-range intracellular transport through coordinated cargo recognition and conformational autoregulation, yet the mechanisms that selectively control cargo engagement remain unclear. Here, we identify a phosphorylation-dependent gate on kinesin-1 activity mediated by the carboxy-terminal domain (CTD) of kinesin light chain 2 (KLC2). The KLC2 CTD is constitutively phosphorylated on multiple serine residues, suppressing membrane association via its amphipathic helix. We identify the NIMA-related kinase NEK10 as a KLC2-selective regulator of kinesin-1, restraining motor activation, cargo engagement, and lysosome transport; conversely, loss of NEK10 increases membrane association and, together with low-affinity adaptor interactions, promotes lysosome motility. These findings reveal a phosphorylation-regulated protein-lipid coincidence-detection mechanism - a kinesin-kinase code - that integrates adaptor binding with membrane cues to control kinesin-1-mediated transport and provide a mechanistic basis for understanding paralogue and isoform diversity in the kinesin-1 family.

cell biology↗

bioRxiv2021

Molecular mechanism for kinesin-1 direct membrane recognition

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxy-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical and cell biology approaches we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a newly defined subset of long kinesin light chain paralogues and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids and is important for kinesin-1 dependent lysosome positioning, a canonical activity that until now has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a new protein-lipid coincidence detection framework for kinesin-1 mediated organelle transport.

cell biology↗

bioRxiv2020

A heterodimeric SNX4:SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly

Efficient mammalian autophagosome biogenesis requires coordinated input from other cellular endomembrane compartments. Such coordination includes the stimulated trafficking to autophagosome assembly sites of the essential autophagy proteins, ATG9 and ATG16L1, via distinct endosomal compartments. Protein trafficking within the endocytic network is directed by a conserved family of sorting nexins (SNXs), with previous studies implicating SNX18 (an SH3 domain-type SNX-BAR protein) in the mobilisation of ATG9A and ATG16L1 from recycling endosomes during autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that a second mammalian SNX-BAR, SNX4, is needed for efficient LC3 lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and heterodimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30, and that these associate with tubulovesicular endocytic membranes at steady state. Detailed image-based analysis during the early stages of autophagosome assembly reveal that SNX4:SNX7 is the autophagy-specific heterodimeric SNX-BAR complex, required for efficient recruitment/retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially co-localises with juxtanuclear ATG9A-positive membranes, with our data linking the SNX4 autophagy defect to the mis-trafficking and/or retention of ATG9A in the Golgi region. Together, our findings show that the SNX4:SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites. SUMMARY STATEMENTA heterodimeric SNX4:SNX7 SNX-BAR complex regulates mammalian autophagosome assembly through the control of ATG9 trafficking.

cell biology↗

bioRxiv2020

Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

Current methodologies for targeting the mitochondrial genome for basic research and/or therapeutic strategy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. The development of a functional molecular toolbox for CRISPR-mediated mitochondrial genome editing is therefore desirable, as this could enable precise targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes; however, published reports of "MitoCRISPR" systems have, to date, lacked reproducibility and independent corroboration. Here, we have explored the requirements for a functional MitoCRISPR system in human cells by engineering several versions of CRISPR nucleases, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of gRNA modifications that reportedly induce mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and influences on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Finally, we present evidence linking mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary if MitoCRISPR is to be realised.

molecular biology↗

bioRxiv2019

LIR-dependent LMX1A/LMX1B autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors in iPSC-derived human mDANs, each contributing to the expression of important autophagy genes including ULK1, ATG7, ATG16L1 and TFEB. Suppression of LMX1A and LMX1B in mDANs reduces basal autophagy, lowers mitochondrial respiration, and elevates mitochondrial ROS levels; meanwhile overexpression protects against rotenone poisoning in mDANs in vitro. Significantly, we show that LMX1A and LMX1B bind to multiple ATG8 proteins via LIR-type interactions, in a manner dependent on subcellular localisation and nutrient status: LMX1B interacts with LC3B in the nucleus under basal conditions via a C-terminal LIR, but binds to cytosolic LC3B and is degraded by autophagy during nutrient starvation, and LIR mutant LMX1B is unable to protect mDANs against rotenone. This establishes an LMX1A/LMX1B-autophagy regulatory nexus that helps explain the protective roles of these transcription factors in the adult midbrain, thus having implications for our understanding of mDAN decline in PD.

cell biology↗