Biology subjects

McKenney, R. J.

Publications and source records attributed to McKenney, R. J..

At least 19 recordsLinked to original sources

bioRxiv2025

Injury-induced tau pathology promotes aggressive behavior in Drosophila without neurodegeneration.

The microtubule-associated protein tau is implicated in neurodegenerative diseases, but its physiological roles remain poorly understood. Here, we find that pan-neuronal expression of human tau (HsTau) in Drosophila coupled with injury triggers hyper-aggression in male flies, which is absent in flies expressing non-phosphorylatable tau. These behavioral manifestations result from activation of dopaminergic circuits without neurodegeneration. Using in vitro reconstitution assays, we find that phosphorylated HsTau maintains microtubule binding but loses its ability to suppress catastrophes, thereby promoting microtubule dynamicity. In contrast, unphosphorylated HsTau and fly tau (DmTau) stabilize microtubules by reducing catastrophe frequency. Our findings challenge the canonical view of tau as a simple microtubule stabilizer and instead position it as a dynamic regulator of microtubule function and neuronal excitability. These results reveal how acute tau phosphorylation can alter neural circuit function and behavior prior to neurodegeneration, providing new insights into taus physiological and pathological roles.

neuroscience↗

bioRxiv2025

Structural Basis of Kinesin-1 Autoinhibition and Its Control of Microtubule-Based Motility

Kinesin-1 was the first identified microtubule-based motor protein that drives anterograde intracellular transport of diverse cargoes in eukaryotic cells. Improper regulation and kinesin-1 defects are implicated in multiple neurological disorders as well as pathogens hijacks kinesin-1 to deliver their cargoes. Despite its importance, the molecular mechanisms governing kinesin-1 regulation and activation remains poorly understood. Here, we report the cryo-EM structure of the autoinhibited kinesin-1 heterotetramer and validate it using crosslinking mass spectrometry. The structure reveals a 36-nm particle in which the kinesin heavy chains (KHCs) adopt a head to tail configuration, stabilized by asymmetrically arranged kinesin light chain (KLC) tetratricopeptide repeat (TPR) domains that bind across folded KHC coiled-coils and wedge in between the KHC motor domains. This architecture inhibits kinesin motility by constraining the dimeric motor domains in a configuration that is incompatible with processive movement. In addition, the structure shows that the KLC C-terminal helices occlude the TPR cargo binding interfaces, revealing a second layer of autoinhibition that directly blocks cargo engagement. Functional studies and structural modeling suggest that binding of regulatory factors, such as MAP7D3, compete with intramolecular KHC coiled-coil interactions, resulting in the unfurling of the autoinhibited structure and activating motor motility. These findings provide a molecular framework for understanding kinesin-1 regulation and its implications for intracellular transport.

biochemistry↗

bioRxiv2025

Microtubule acetylation is a biomarker of cytoplasmic health during cellular senescence

Cellular senescence is marked by cytoskeletal dysfunction, yet the role of microtubule post-translational modifications (PTMs) remains unclear. We demonstrate that microtubule acetylation increases during drug-induced senescence in human cells and during natural aging in Drosophila. Elevating acetylation via HDAC6 inhibition or TAT1 overexpression in BEAS-2B cells disrupts anterograde Rab6A vesicle transport, but spares retrograde transport of Rab5 endosomes. Hyperacetylation results in slowed microtubule polymerization and decreased cytoplasmic fluidity, impeding diffusion of micron-sized condensates. These effects are distinct from enhanced detyrosination, and correlate with altered viscoelasticity and resistance to osmotic stress. Modulating cytoplasmic viscosity reciprocally perturbs microtubule dynamics, revealing bidirectional mechanical regulation. Senescent cells phenocopy hyperacetylated cells, exhibiting analogous effects on transport and microtubule polymerization. Our findings establish acetylation as a biomarker for cytoplasmic health and a potential driver of age-related cytoplasmic densification and organelle transport decline, linking microtubule PTMs to biomechanical feedback loops that exacerbate senescence. This work highlights the role of acetylation in bridging cytoskeletal changes to broader aging hallmarks.

cell biology↗

bioRxiv2025

The KASH protein UNC-83 differentially regulates kinesin-1 activity to control developmental stage-specific nuclear migration

Nuclear migration plays a fundamental role in development, requiring precise spatiotemporal control of bidirectional movement through dynein and kinesin motors. Here, we uncover a mechanism for developmental regulation of nuclear migration directionality. The nuclear envelope KASH protein UNC-83 in Caenorhabditis elegans exists in multiple isoforms that differentially control motor activity. The shorter UNC-83c isoform promotes kinesin-1-dependent nuclear movement in embryonic hyp7 precursors, while longer UNC-83a/b isoforms facilitate dynein-mediated nuclear migration in larval P cells. We demonstrate that UNC-83as N-terminal domain functions as a kinesin-1 inhibitory module by directly binding kinesin heavy chain (UNC-116). This isoform-specific inhibition, combined with differential affinity for kinesin light chain (KLC-2), establishes a molecular switch for directional control. Together, these interdisciplinary studies reveal how alternative isoforms of cargo adaptors can generate developmental stage-specific regulation of motor activity during development.

cell biology↗

bioRxiv2025

A pathological phosphorylation pattern enhances taucooperativity on microtubules and facilitates tau filamentassembly

Phosphorylation plays a crucial role in both normal and disease processes involving the microtubule-associated protein tau. Physiologically, phosphorylation regulates taus subcellular localization within neurons and is involved in fetal development and animal hibernation. However, abnormal phosphorylation of tau is linked to the formation of neurofibrillary tangles (NFTs) in various human tauopathies. Interestingly, the patterns of tau phosphorylation are similar in both normal and abnormal processes, leaving unclear whether phosphorylated tau retains its functional role in normal processes. The relationship between tau phosphorylation and NFT assembly in tauopathies is also still debated. To address these questions, we investigated the effects of tau phosphorylation on microtubule binding, cooperative protein envelope formation, and NFT filament assembly relevant to tauopathies. Consistent with previous results, our findings show that tau phosphorylation decreases taus overall affinity for microtubules, but we reveal that phosphorylation more dramatically impacts the cooperativity between tau molecules during tau envelope formation along microtubules. Additionally, we observed that the specific pattern of phosphorylation, rather than overall phosphorylation level, strongly impacts the assembly of tau filaments in vitro. Our results reveal new insights into how tau phosphorylation impacts taus physiological roles on microtubules and its pathoconversion into NFTs.

biochemistry↗

bioRxiv2025

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

Cytoplasmic dynein is an essential microtubule motor protein that powers organelle transport and mitotic spindle assembly. Its activity depends on dynein-dynactin-cargo adaptor complexes, such as dynein-dynactin-BicD2 (DDB), which typically function with two dynein motors. We show that mechanical tension recruits a third dynein motor via an auxiliary BicD adaptor binding the light intermediate chain of the third dynein, stabilizing multi-dynein assemblies and enhancing force generation. Lis1 prevents dynein from transitioning into a force-limiting phi-like conformation, allowing single-dynein DDB to sustain forces up to [~]4.5 pN, whereas force generation often ends at [~]2.5 pN without Lis1. Complexes with two or three dyneins generate [~]7 pN and [~]9 pN, respectively, consistent with a staggered motor arrangement that enhances collective output. Under load, DDB primarily takes [~]8 nm steps, challenging existing dynein coordination models. These findings reveal adaptive mechanisms that enable robust intracellular transport under varying mechanical demands.

biophysics↗

bioRxiv2024

Active Microtubule-Actin Crosstalk Mediated by a Nesprin-2G-Kinesin Complex

Nesprins are integral membrane proteins that physically couple the nucleus and cytoskeleton. Nesprin-2 Giant (N2G) stands out for its extensive cytoplasmic domain, which contains tandem N-terminal actin-binding calponin-homology domains followed by >50 spectrin repeats and a C-terminal outer nuclear membrane-spanning KASH domain. N2Gs KASH domain interacts with the inner nuclear membrane, lamina-binding SUN proteins within the perinuclear space, forming a linker of nucleoskeleton and cytoskeleton (LINC) complex. Additionally, N2G contains a conserved W-acidic LEWD motif that enables the direct interaction with kinesin-1s light chain, indicating N2Gs involvement with both actin and microtubules. The absence of N2G leads to embryonic lethality in mice, while cellular assays highlight N2Gs role in nuclear positioning across diverse biological contexts. However, the precise mechanisms underlying N2G-mediated nucleocytoskeletal coupling remain unclear. Here we study N2Gs interactions with F-actin and kinesin-1, revealing its functions as an F-actin bundler, a kinesin-1-activating adapter, and a mediator of active cytoskeletal crosstalk. Along with MAP7 proteins, N2G directly links active kinesin-1 motors to F-actin, facilitating actin transport along microtubule tracks. These findings shed light on N2Gs dynamic role as a crosslinker between actin and microtubule cytoskeletons, offering insights into nuclear movement, a fundamental cellular process.

biophysics↗

bioRxiv2023

Conserved Roles for the Dynein Intermediate Chain and Ndel1 in Assembly and Activation of Dynein

Cytoplasmic dynein, the primary retrograde microtubule transport motor within cells, must be activated for processive motility through the regulated assembly of a dynein-dynactin-adapter (DDA) complex. The interaction between dynein and dynactin was initially ascribed to the N-terminus of the dynein intermediate chain (IC) and a coiled-coil of the dynactin subunit p150Glued. However, cryo-EM structures of DDA complexes have not resolve these regions of the IC and p150Glued, raising questions about the importance of this interaction. The IC N-terminus (ICN) also interacts with the dynein regulators Nde1/Ndel1, which compete with p150Glued for binding to ICN. Using a combination of approaches, we reveal that the ICN plays critical, evolutionarily conserved roles in DDA assembly by interacting with dynactin and Ndel1, the latter of which recruits the DDA assembly factor LIS1 to the dynein complex. In contrast to prior models, we find that LIS1 cannot simultaneously bind to Ndel1 and dynein, indicating that LIS1 must be handed off from Ndel1 to dynein in temporally discrete steps. Whereas exogenous Ndel1 or p150Glued disrupts DDA complex assembly in vitro, neither perturbs preassembled DDA complexes, indicating that the IC is stably bound to p150Glued within activated DDA complexes. Our study reveals previously unknown regulatory steps in the dynein activation pathway, and provides a more complete model for how the activities of LIS1/Ndel1 and dynactin/cargo-adapters are integrated to regulate dynein motor activity.

biochemistry↗

bioRxiv2022

Differential requirement for BRCA1-BARD1 E3 ubiquitin ligase activity in DNA damage repair and meiosis in the Caenorhabditis elegans germ line

The tumor suppressor BRCA1-BARD1 complex functions in many cellular processes; of critical importance to its tumor suppressor function is its role in genome integrity. Although RING E3 ubiquitin ligase activity is the only known enzymatic activity of the complex, the in vivo requirement for BRCA1-BARD1 E3 ubiquitin ligase activity has been controversial. Here we probe the role of BRCA1-BARD1 E3 ubiquitin ligase activity in vivo using C. elegans. Genetic, cell biological, and biochemical analyses of mutants defective for E3 ligase activity reveal both E3 ligase-dependent and independent functions of the complex in the context of DNA damage repair and meiosis. We show that E3 ligase activity is essential for BRCA1-BARD1 to concentrate at both DNA damage and recombination sites in meiotic germ cells, but not at DNA damage sites in proliferating germ cells. While BRCA1 alone is capable of monoubiquitylation, BARD1 is required with BRCA1 to promote polyubiquitylation. We find that the requirement for E3 ligase activity and BARD1 in DNA damage signaling and repair can be partially alleviated by driving the nuclear accumulation and self-association of BRCA1. Our data suggest that in addition to E3 ligase activity, BRC-1 serves a structural role for DNA damage signaling and repair while BRD-1 plays an accessory role to enhance BRC-1 function. Author SummaryBRCA1-BARD1 is a E3 ubiquitin ligase, which modifies proteins by the addition of the small protein ubiquitin. While mutations that disrupt E3 ligase activity and stability of the BRCA1-BARD1 complex lead to a predisposition for breast and ovarian cancer, the specific requirement for E3 ligase activity in tumor suppression is not known. Here we probe the function of E3 ligase activity and BARD1 in the maintenance of genome integrity by engineering point mutations that disrupt E3 ligase activity in C. elegans BRCA1 as well as a null mutation in BARD1. We find that while E3 ligase activity is important for genome integrity, the complex plays additional roles besides ubiquitylating proteins. Further, our data suggest that BRCA1 is the key functional unit of the complex while BARD1 is an accessory partner that enhances BRCA1s function. These findings may help explain why there is a higher prevalence of cancer-causing mutations in BRCA1 compared to BARD1.

genetics↗

bioRxiv2022

Control of Motor Landing and Processivity by the CAP-Gly Domain in the KIF13B Tail

Microtubules are a major component of the eukaryotic cytoskeleton that play crucial roles in diverse cellular process. Posttranslational modifications (PTMs) of tubulin dimers regulate the dynamics and organization of microtubules, as well as the interactions between microtubules and microtubule-associated proteins (MAPs). One unique PTM that occurs on microtubules is the cyclical removal and re-addition of the C-terminal tyrosine of -tubulin. CAP-Gly (cytoskeleton-associated protein glycine-rich) domain containing proteins specifically recognize tyrosinated microtubules, a property exploited to regulate and spatially localize diverse microtubule effectors. KIF13B is a member of the long-distance transport kinesin-3 family, and the only kinesin motor that contains a conserved C-terminal CAP-Gly domain. What role the CAP-Gly domain plays in KIF13Bs motility along microtubules is unknown. Here, we investigated the interaction between KIF13Bs CAP-Gly domain, and the tyrosinated C-terminal tail domain of -tubulin. We found that KIF13Bs CAP-Gly domain strongly influences the initial motor-microtubule interaction, as well as the processive motility of KIF13B along microtubules. The effect of the CAP-Gly domain on kinesin-microtubule binding is specific to the nucleotide state of the motor domain, suggesting an interplay between the N-terminal motor domain and C-terminal CAP-Gly domain underlies the KIF13B-microtubule interaction. These results reveal that specialized kinesin tail domains play active roles in the initiation and continuation of motor movement.

biophysics↗

bioRxiv2022

The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit

The Ndc80 kinetochore complex is essential for robust kinetochore-microtubule (k-MT) attachments during mitosis. Ndc80 has been shown to recruit the Ska1 complex to kinetochores, where Ska1 is thought to aid in k-MT coupling by Ndc80. Our previous work has shown that Cdt1, a DNA replication licensing factor, is a novel mitotic spindle-associated protein that is also recruited to kinetochores via Ndc80 and is required for stabilizing k-MT attachments. In this study, we developed auxin-induced degron (AID)-tagging to validate the previously demonstrated mitotic role of Cdt1. We demonstrate a direct interaction between Cdt1 and Ska1 that is essential for proper recruitment of Cdt1 to kinetochores and spindle microtubules. We find that Cdk1dependent phosphorylation of Cdt1 during mitosis is critical for Ska1-binding, consequently regulating the stabilization of metaphase k-MT attachments and normal mitotic progression. Total internal reflection fluorescence microscopy (TIR-FM) experiments reveal that Cdt1 synergizes with the Ndc80 and the Ska1 complexes for microtubule-binding. Further, we show that single Cdt1 molecules form diffusive tripartite complexes with Ndc80 and Ska1 that can processively track the ends of dynamic microtubules in vitro. Taken together our data identifies a minimal molecular unit responsible for bidirectional processive tip tracking of kinetochores.

cell biology↗

bioRxiv2021

Microtubule Lattice Spacing Governs Cohesive Envelope Formation of Tau Family Proteins

Tau is an intrinsically-disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive "envelopes" around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here, we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau-envelope disassembly. Investigating other members of the tau-MAP family, we find MAP2 similarly forms envelopes governed by lattice-spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct segments. We conclude that the interdependent allostery between lattice-spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

biophysics↗

bioRxiv2021

Reconstitution of Kinesin-1 Activation

Autoinhibition is an important regulatory mechanism for cytoskeletal motor proteins. Kinesin-1 (kinesin hereafter), the ubiquitous plus-end directed microtubule motor, is thought to be controlled by a complicated autoinihibition mechanism, but the molecular details remain unclear. Conformational changes mediated by intramolecular interactions between the C-terminal tail and N-terminal motor domains of the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The dimeric KHC also binds two copies of the kinesin light chains (KLCs), which have been implicated in both autoinhibition and cargo-dependent activation of the tetrameric motor complex, although the precise mechanisms remain opaque. Using in vitro reconstitution, we show that the KLC strongly inhibits the kinesin-microtubule interaction via an independent mechanism from the tail-motor interaction within KHC. Kinesin cargo-adaptor proteins that bind KLC activated processive movement of the kinesin tetramer but the landing rate of these activated complexes remained low. The addition of MAP7, which specifically binds to the KHC, strongly enhanced activated motor motility by dramatically increasing the landing rate and processivity of the activated kinesin motors. Our results support a model whereby the activity of the kinesin tetramer is regulated by independent tail- and KLC-based inhibition mechanisms, and that cargo-adaptor binding to the KLC directly releases both of these inhibitions. However, we find that a third component, a non-motor MAP is required for robust activity of the activated motor. Thus, human kinesin activity is regulated by a two-factor mechanism comprised of intramolecular allosteric regulation, as well as intermolecular kinesin-adaptor and kinesin-MAP interactions.

biophysics↗

bioRxiv2020

Structural stability of SARS-CoV-2 degrades with temperature

SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter. Statement of Scientific SignificanceThe economic and public health impact of the COVID-19 pandemic are very significant. However scientific information needed to underpin policy decisions are limited partly due to novelty of the SARS-CoV-2 pathogen. There is therefore an urgent need for mechanistic studies of both COVID-19 disease and the SARS-CoV-2 virus. We show that individual virus particles suffer structural destabilization at relatively mild but elevated temperatures. Our nanoscale results are consistent with recent observations at larger scales. Our work strengthens the case for COVID-19 resurgence in winter.

biophysics↗

bioRxiv2020

A Highly Conserved 310-Helix Within the Kinesin Motor Domain is Critical for Kinesin Function and Human Health.

KIF1A, a kinesin-3 family member, plays critical roles as a long-distance cargo-transporter within neurons. Over 100 known KIF1A mutations in humans result in KIF1A Associated Neurological Disease (KAND), developmental and degenerative neurological conditions for which there is no cure. A de novo missense mutation, P305L, was recently identified in several children diagnosed with KAND, but the underlying molecular basis for the disease phenotype is unknown. Interestingly, this residue is highly conserved in kinesin-family proteins, and together with adjacent conserved residues also implicated in KAND, forms an unusual 310-helical element immediately C-terminal to loop-12 (L12, also known as the K-loop in KIF1A) in the conserved kinesin motor core. In KIF1A, the disordered K-loop contains a highly charged insertion of lysines that is thought to endow the motor with a high microtubule-association rate. Here, we characterize the molecular defects of the P305L mutation in KIF1A using genetic, biochemical, and single-molecule approaches. We find the mutation negatively impacts the velocity, run-length, and force generation of the motor. However, a much more dramatic effect is observed on the microtubule-association rate of the motor, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that an elusive K-loop conformation, mediated by formation of a highly conserved adjacent 310-helix that is modulated via P305, is critically important for the kinesin-microtubule interaction. Importantly, we find that the function of this proline is conserved in the canonical kinesin, KIF5, revealing a fundamental principle of the kinesin motor mechanism.

biophysics↗

bioRxiv2019

A Combinatorial MAP Code Dictates Polarized Microtubule Transport

Many eukaryotic cells distribute their intracellular components through asymmetrically regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that these MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors, and reveal how transient interactions between MAPs and motors may promote these effects. From these data, we propose a general \"MAP code\" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.

biophysics↗

bioRxiv2019

Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors

KIF1A is a kinesin-family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules. In humans, more than ten point mutations in KIF1A are associated with the motor neuron disease, hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus, a clear molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the pure form of spastic paraplegia hyperactivate motility of the KIF1A motor. Introduction of the corresponding mutations into Caenorhabditis elegans KIF1A homologue unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyper-activation of kinesin motor activity, rather than its loss-of-function, is a novel cause of motor neuron disease in humans.\n\nSignificance StatementAnterograde axonal transport supplies organelles and protein complexes throughout axonal processes to support neuronal morphology and function. It has been observed that reduced anterograde axonal transport is associated with neuronal diseases. In contrast, here we show that particular disease-associated mutations in KIF1A, an anterograde axonal motor for synaptic vesicle precursors, induce hyperactivation of KIF1A motor activity and increased axonal transport of synaptic vesicle precursors. Our results advance the knowledge of the regulation of motor proteins and axonal transport and cell biology of motor neuron diseases.

cell biology↗

bioRxiv2018

Microtubules Gate Tau Condensation to Spatially Regulate Microtubule Functions

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimers disease and other dementias, yet the physiological state of tau molecules within cells remains unclear. Using single molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of MT severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, controlled by the microtubule, is an important mechanism of taus biological functions, and that oligomerization of tau is a common property shared between the physiological and disease forms of the molecule.\n\nOne Sentence SummaryReversible tau oligomerization regulates microtubule functions.

biophysics↗