Twelvetrees Lab

Dynein-driven regulation of postsynaptic membrane architecture and synaptic function […]

tttrlib: modular software for integrating fluorescence spectroscopy, imaging, and molecular modeling […]

The Power of Three: Dynactin associates with three dyneins under load for greater force production […]

Biocompatible sulfonium-based covalent probes for endogenous tubulin fluorescence nanoscopy in live and fixed cells [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2025.01.27.635008v1?rss=1

Favorable response to ketogenic diet therapy in a patient with DYNC1H1-related epilepsy […]

Two cases of Perry disease (Perry syndrome) in the same family with normal 123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy […]

Caliber of zebrafish touch-sensory axons is dynamic in vivo [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2024.12.04.626901v2?rss=1

A Two-Heads-Bound State Drives KIF1A Superprocessivity - PubMed 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 …

A Two-Heads-Bound State Drives KIF1A Superprocessivity
https://pubmed.ncbi.nlm.nih.gov/39868206/?utm_source=Inoreader&utm_medium=rss&utm_campaign=pubmed-2&utm_content=1ZcXDdiuMGSdBND0i1xMoc0h12MBMQBJF0eflC9OhMFnQBfEJ_&fc=20230717091213&ff=20250127144029&v=2.18.0.post9+e462414

Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT […]

Axon Trafficking Counteracts Aberrant Protein Aggregation in Neurons [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2025.01.16.633295v1?rss=1

Auxin Triggers AHR Pathway Activation in the Auxin-Inducible Degron System in Mammalian Cells […]

A novel approach to tagging tubulin reveals microtubule assembly dynamics of the axoneme in Trypanosoma brucei [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2025.01.27.634986v1?rss=1

Cytoplasmic streaming of symbiotic algae in the ciliate Stentor pyriformis
https://pubmed.ncbi.nlm.nih.gov/39864254/?utm_source=Inoreader&utm_medium=rss&utm_campaign=pubmed-2&utm_content=1ZcXDdiuMGSdBND0i1xMoc0h12MBMQBJF0eflC9OhMFnQBfEJ_&fc=20230717091213&ff=20250127022952&v=2.18.0.post9+e462414

BicD and MAP7 collaborate to activate homodimeric Drosophila kinesin-1 by complementary mechanisms […]

Impact of physiological ionic strength and crowding on kinesin-1 motility - PubMed 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 …

Impact of physiological ionic strength and crowding on kinesin-1 motility
https://pubmed.ncbi.nlm.nih.gov/39779244/?utm_source=Inoreader&utm_medium=rss&utm_campaign=pubmed-2&utm_content=1ZcXDdiuMGSdBND0i1xMoc0h12MBMQBJF0eflC9OhMFnQBfEJ_&fc=20230717091213&ff=20250109032023&v=2.18.0.post9+e462414

TDP-43 Cryptic RNAs in Perry Syndrome: Differences across Brain Regions and TDP-43 Proteinopathies […]

ATG9 vesicles are a subtype of intracellular nanovesicle 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.

ATG9 vesicles are a subtype of intracellular nanovesicle [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2024.09.12.612637v2?rss=1

Super-photostable organic dye for long-term live-cell single-protein imaging - PubMed 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 …

Super-photostable organic dye for long-term live-cell single-protein imaging
https://pubmed.ncbi.nlm.nih.gov/39815105/?utm_source=Inoreader&utm_medium=rss&utm_campaign=pubmed-2&utm_content=1ngYFTZ0OsNq0jYw1SOAw0IMOsFnD7i7eNYr2_u4fqrfaND9Zs&fc=20230717091338&ff=20250116024945&v=2.18.0.post9+e462414

Elongator is a microtubule polymerase selective for polyglutamylated tubulin
https://www.embopress.org/doi/10.1038/s44318-024-00358-0?af=R

Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport 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.

Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2024.08.30.610563v2?rss=1

Technical and biological sources of noise confound multiplexed enhancer AAV screening 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.

Technical and biological sources of noise confound multiplexed enhancer AAV screening [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2025.01.14.633018v1?rss=1

BicD and MAP7 collaborate to activate homodimeric Drosophila kinesin-1 by complementary mechanisms 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.

BicD and MAP7 collaborate to activate homodimeric Drosophila kinesin-1 by complementary mechanisms
http://biorxiv.org/cgi/content/short/2025.01.11.632512v1?rss=1

β3 accelerates microtubule plus end maturation through a divergent lateral interface
https://www.molbiolcell.org/doi/abs/10.1091/mbc.E24-08-0354?af=R

Single-cell RNA sequencing across isogenic FUS and TARDBP ALS lines reveals a shared early mitochondrial dysfunction unique to motor neurons 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

Single-cell RNA sequencing across isogenic FUS and TARDBP ALS lines reveals a shared early mitochondrial dysfunction unique to motor neurons. [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2023.03.16.531876v3?rss=1

Systematic Identification of Microtubule Inner Proteins Reveals JPT2 as a Key Regulator of Lumen Microenvironment and Drug Sensitivity 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.

Systematic Identification of Microtubule Inner Proteins Reveals JPT2 as a Key Regulator of Lumen Microenvironment and Drug Sensitivity [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2025.01.12.632577v1?rss=1

Mechanistic basis of temperature adaptation in microtubule dynamics across frog species
https://www.cell.com/current-biology/fulltext/S0960-9822(24)01690-7?rss=yes

Oversized cells activate global proteasome-mediated protein degradation to maintain cell size homeostasis

Oversized cells activate global proteasome-mediated protein degradation to maintain cell size homeostasis
https://elifesciences.org/articles/75393

KIF1C activates and extends dynein movement through the FHF cargo adapter - Nature Structural & Molecular Biology Here the authors show that codependence of dynein and kinesin KIF1C occurs through binding of the FTS–HOOK3–FHIP1B cargo adapter. Binding of KIF1C releases the HOOK3 autoinhibited folded conformation allowing dynein to bind the adapter. In this cocomplex, KIF1C further acts as a processivity factor for dynein.

KIF1C activates and extends dynein movement through the FHF cargo adapter
https://www.nature.com/articles/s41594-024-01418-z

AXONAL distribution of mitochondria maintains neuronal autophagy during aging via eIF2β Neuronal aging and neurodegenerative diseases are accompanied by proteostasis collapse, while cellular factors that trigger it are not identified. Impaired mitochondrial transport in the axon is another feature of aging and neurodegenerative diseases. Using Drosophila , we found that genetic depletion of axonal mitochondria causes dysregulation of protein degradation. Axons with mitochondrial depletion showed abnormal protein accumulation and autophagic defects. Lowering neuronal ATP levels by blocking glycolysis did not reduce autophagy, suggesting that autophagic defects are associated with mitochondrial distribution. We found that eIF2β was increased by the depletion of axonal mitochondria via proteome analysis. Phosphorylation of eIF2α, another subunit of eIF2, was lowered, and global translation was suppressed. Neuronal overexpression of eIF2β phenocopied the autophagic defects and neuronal dysfunctions, and lowering eIF2β expression rescued those perturbations caused by depletion of axonal mitochondria. These results indicate the mitochondria-eIF2β axis maintains proteostasis in the axon, of which disruption may underly the onset and progression of age-related neurodegenerative diseases. Highlights ### Competing Interest Statement The authors have declared no competing interest. The datasets used and/or analyzed during the current study are available from the corresponding author upon request.

AXONAL distribution of mitochondria maintains neuronal autophagy during aging via eIF2{beta} [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2024.01.20.576435v2?rss=1

Different mechanisms link gain and loss of kinesin functions to axonal degeneration Axons are the slender, often meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organism’s lifetime, meaning up to a century in humans. Long-term maintenance and sustained functionality of axons requires motor protein-driven transport distributing life-sustaining materials and organelles to places of need. It seems therefore plausible that loss of motor function can cause axon degeneration; however, also gain-of-function conditions were linked to disorders including motor neuron disease or spastic paraplegia. To understand this phenomenon, we studied ∼40 genetic manipulations of motor proteins, cargo linkers and regulators of reactive oxygen species in one standardised Drosophila primary neuron system. Using axonal microtubule bundle organisation as a relevant readout reflecting the state of axon integrity, we found that losses of Dynein heavy chain, KIF1A/Unc-104 and KIF5/Kinesin heavy chain (Khc) all cause bundle disintegration in the form of chaotically curled microtubules. Detailed functional studies of Khc and its adaptor proteins revealed that losses of mitochondrial or lysosomal transport cause ROS dyshomeostasis, which is a condition that inducing MT-curling in fly and mouse neurons alike. We find that hyper-activated Khc induces the same MT-curling phenotype, not through ROS but directly through enhanced mechanical forces. Studies with loss of Unc-104 and expression of an ALS-linked mutant form of the human Khc orthologue KIF5A suggest that the two mechanisms apply to motors beyond Khc. We discuss a model which can explain the surprising common outcome of both conditions and examine its relevance for understanding motor-linked neurodegeneration. ### Competing Interest Statement The authors have declared no competing interest.

Different mechanisms link gain and loss of kinesin functions to axonal degeneration [NEW RESULTS]
http://biorxiv.org/cgi/content/short/2024.12.31.630930v1?rss=1