KIF1A, a neuron-specific Kinesin-3 motor, is indispensable for long-distance axonal transport and nuclear migration, processes vital for neuronal function. Using MINFLUX tracking, we reveal that KIF1A predominantly adopts a two-heads-bound state, even under ATP-limiting conditions, challenging prior …

The motility of biological molecular motors has typically been analyzed by in vitro reconstitution systems using motors isolated and purified from organs or expressed in cultured cells. The behavior of biomolecular motors within cells has frequently been reported to be inconsistent with that observe …

Cells are filled with thousands of vesicles, which mediate protein transport and ensure homeostasis of the endomembrane system. Distinguishing these vesicles functionally and molecularly represents a major challenge. Intracellular nanovesicles (INVs) are a large class of transport vesicles that likely comprises of multiple subtypes. Here, we define the INV proteome and find that it is molecularly heterogeneous, and enriched for transmembrane cargo molecules including integrins, transporters, and ATG9A, a lipid scramblase associated with autophagy. ATG9A is known to reside in ‘ATG9 vesicles’: small vesicles that contribute to autophagosome formation. Using in-cell vesicle capture assays we found that ATG9A, as well as other ATG9 vesicle cargos, were in INVs. Quantitative analysis showed that virtually all ATG9 vesicles are INVs, but that only ∼20% of INVs are ATG9 vesicles, suggesting that ATG9 vesicles are in fact a subtype of INV, which we term ATG9A-flavor INVs. Finally, we show that perturbing ATG9A-flavor INVs impaired the autophagy response induced by starvation. ### Competing Interest Statement The authors have declared no competing interest.

Organic dyes play a crucial role in live-cell imaging because of their advantageous properties, such as photostability and high brightness. Here we introduce a super-photostable and bright organic dye, Phoenix Fluor 555 (PF555), which exhibits an order-of-magnitude longer photobleaching lifetime tha …

Programmed ribosomal frameshifting is a process where a proportion of ribosomes change their reading frame on an mRNA, rephasing the ribosome relative to the mRNA. While frameshifting is commonly employed by viruses, very few phylogenetically conserved examples are known in nuclear encoded genes and some of the evidence is controversial. Here we report a +1 frameshifting event during decoding of the human gene PLEKHM2. This frameshifting occurs at the sequence UCC\_UUU\_CGG, which is conserved in vertebrates and is similar to an influenza virus sequence that frameshifts with similar efficiency. The new C-terminal domain generated by this frameshift forms an α-helix, which relieves PLEKHM2 from autoinhibition and allows it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Reintroducing both the canonically-translated and frameshifted protein are necessary to restore normal contractile function of PLEKHM2-knockout cardiomyocytes, demonstrating the necessity of frameshifting for normal cardiac activity. ### Competing Interest Statement The authors have declared no competing interest.

Cis-acting regulatory enhancer elements are valuable tools for gaining cell type-specific genetic access. Leveraging large chromatin accessibility atlases, putative enhancer sequences can be identified and deployed in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck in enhancer AAV discovery is charting their detailed expression patterns in vivo, a process that currently requires gold-standard one-by-one testing. Here we present a barcoded multiplex strategy for screening enhancer AAVs at cell type resolution using single cell RNA sequencing and taxonomy mapping. We executed a proof-of-concept study using a small pool of validated enhancer AAVs expressing in a variety of neuronal and non-neuronal cell types across the mouse brain. Unexpectedly, we encountered substantial technical and biological noise including chimeric packaging products, necessitating development of novel techniques to accurately deconvolve enhancer expression patterns. These results underscore the need for improved methods to mitigate noise and highlight the complexity of enhancer AAV biology in vivo. ### Competing Interest Statement Authors JTT, BPL, BT, ESL, JKM are co-inventors on patent application PCT/US2021/45995 Artificial expression constructs for selectively modulating gene expression in striatal neurons. Authors JTT, BPL, BT are co-inventors on provisional patent application US 63/582,759 Artificial expression constructs for modulating gene expression in the basal ganglia. JKM and BPL declare equity in EpiCure Therapeutics, Inc. JS is a scientific advisory board member, consultant and/or co-founder of Cajal Neuroscience, Guardant Health, Maze Therapeutics, Camp4 Therapeutics, Phase Genomics, Adaptive Biotechnologies, Scale Biosciences, Sixth Street Capital, Prime Medicine, Somite Therapeutics and Pacific Biosciences.

The folded auto-inhibited state of kinesin-1 is stabilized by multiple weak interactions and binds weakly to microtubules. Here we investigate the extent to which homodimeric Drosophila kinesin-1 lacking light chains is activated by the dynein activating adaptor Drosophila BicD. We show that one or two kinesins can bind to the central region of BicD (CC2), a region distinct from that which binds dynein-dynactin (CC1) and cargo-adaptor proteins (CC3). Kinesin light chain significantly reduces the amount of kinesin bound to BicD and thus regulates this interaction. Binding of kinesin to BicD increases the number of motors bound to the microtubule, the fraction moving processively and the run length, suggesting that BicD relieves kinesin auto-inhibition. In contrast, microtubule-associated protein 7 (MAP7) has minimal impact on the percentage of motors moving processively but enhances both kinesin-1 recruitment to microtubules and run length. BicD relieves auto-inhibition of kinesin, while MAP7 enables activated motors to engage productively with microtubules. When BicD and MAP7 are combined, the most robust activation of kinesin-1 occurs, highlighting the crosstalk between adaptors and microtubule associated proteins in regulating transport. These observations imply that when both dynein and kinesin-1 are simultaneously bound to BicD, the direction the complex moves on MTs will be influenced by MAP7 and the number of bound kinesins. ### Competing Interest Statement The authors have declared no competing interest.

Mutations in the RNA/DNA-binding proteins FUS and TDP-43 cause the fatal disease amyotrophic lateral sclerosis (ALS). The precise mechanisms behind the selective motor neuron degeneration remain unclear and it is uncertain if ALS-causative mutations trigger motor neuron death through shared or distinct pathogenic pathways. To address these two questions, we performed single-cell RNA sequencing across neuron types derived from isogenic induced pluripotent stem cell lines, harbouring FUS P525L, FUS R495X, TARDBP M337V mutations or FUS knockout. The mutations elicited 5- to 15-fold greater transcriptional responses in motor neurons than interneurons. Approximately 20% of transcripts uniquely dysregulated in motor neurons were shared across FUS mutations, with half being driven by FUS gain-of-function. Among these, a majority pointed towards mitochondrial impairments, with attenuated pathways shared with the TARDBP M337V mutation. Meta-analysis demonstrated convergence on mitochondrial dysfunction with C9orf72 -ALS patient-derived motor neurons. We observed impaired mitochondrial motility across ALS motor axons, even in isogenic FUS R244C motor neurons, which retain FUS in the nucleus, demonstrating shared toxic gain-of-function mechanisms across FUS - and TARDBP -ALS, uncoupled from protein mislocalization. These early signs of mitochondrial dysfunction unique to motor neurons could have profound implications for their survival and represent promising therapeutic targets across multiple ALS forms. ### Competing Interest Statement The authors have declared no competing interest. * ALS : amyotrophic lateral sclerosis ANOVA : analysis of variance ATP : adenosine triphosphate 2-DG : 2-deoxyglucose CCA : canonical correlation analysis DEA : differential (gene) expression analysis DEG : differentially expressed genes DNP : 2,4-dinitrophenol DTT : dithiothreitol ECAR : extracellular acidification rate FDR : false discovery rate GO : gene ontology GOF : gain of function GSEA : gene set enrichment analysis HRE : hexanucleotide repeat expansion (in C9orf72) IPA : Ingenuity pathway analysis iPSC : induced pluripotent stem cells KO : knockout LOF : loss of function NLS : nuclear localization signal OCR : oxygen consumption rate PCR : polymerase chain reaction qPCR : quantitative PCR RPKM : reads per kilobase of exon per million reads mapped SEM : standard error of the mean UMAP : uniform manifold approximation and projection

Microtubules are primarily studied for the interactions of proteins that bind to their outer surfaces and ends, while the regulatory mechanisms within the microtubule lumen, particularly in singlet microtubules critical for essential cellular processes, remain largely unexplored. Our study provides the first systematic identification of key regulatory proteins within the single microtubule lumen. Using proximity-dependent biotin identification (Bio-ID) coupled with mass spectrometry, we identified candidate microtubule inner proteins (MIPs), including Jupiter microtubule-associated homolog 2 (JPT2). JPT2 binds directly to microtubules and specifically localizes within the lumen, where it modulates the luminal environment by inhibiting acetylase MEC17 and independently affects the binding and efficacy of Paclitaxel. Furthermore, our screening identified additional MIPs that influence cellular sensitivity to Paclitaxel, indicating a link between luminal regulation and drug responsiveness. These discoveries reveal JPT2’s critical role in singlet microtubule regulation and suggest new therapeutic targets for enhancing cancer drug sensitivity. ### Competing Interest Statement The authors have declared no competing interest.