Biology subjects

Gillies, J. P.

Publications and source records attributed to Gillies, J. P..

9 recordsLinked to original sources

bioRxiv2025

Occludin Acts as a Dynein Adaptor Regulating Permeability and Collateral Angiogenesis

Previous studies of the tight junction protein occludin (OCLN) suggest that multiple phosphorylation sites on the carboxy-terminal domain contribute a regulatory role in vascular barrier properties. However, gene deletion studies failed to identify a clear functional role for OCLN, despite multiple phenotypic alterations. Importantly, previous studies targeting exon 3 allowed expression of a splice variant starting at exon 4, (isoform 4) that expresses the full carboxy-terminal tail. Here we show that the OCLN carboxy-terminus forms a complex with the light intermediate chain (LIC) of dynein to link tight junction cargo to the minus end directed motor protein. Mutational analysis revealed S471 phosphorylation is required for binding to the LIC while S490 phosphorylation is required for trafficking. Expressing OCLN S490A mutant prevented endothelial cell proliferation and collateral angiogenesis. OCLN gene deletion targeting exon 5, preventing full length and isoform 4 expression, resulted in embryonic lethality. In summary, OCLN links tight junction cargo to the dynein motor, regulating trafficking in a phosphorylation dependent manner and contributing to both VEGF-induced vascular permeability and collateral angiogenesis.

cell biology↗

bioRxiv2025

Synthetic cargo adaptors reveal molecular features that can enhance dynein activation

Cytoplasmic dynein-1 (dynein) facilitates the microtubule-based retrograde trafficking of all cellular cargo. To become active, dynein binds dynactin and one of many cargo-specific adaptors to form the active transport complex. Despite having similar structures, active transport complexes assembled with different adaptors move with different properties in vitro. To explore how adaptors differentially activate dynein, we engineered a library of synthetic adaptors and characterized their ability to activate dynein using in vitro reconstitution and cell-based trafficking assays. We found that the apparent motility of dynein is highly plastic and tunable by the adaptor sequence and that it is possible to engineer adaptors that outperform endogenous adaptors ability to generate highly motile active transport complexes. We also found that different adaptors support distinct trafficking behavior and cargo movement in cells. These findings provide insight into how dynein motility is modulated to meet the unique trafficking requirements of all cellular cargo.

biophysics↗

bioRxiv2024

Cargo adaptor identity controls the mechanism and kinetics of dynein activation

Cytoplasmic dynein-1 (dynein), the primary retrograde motor in most eukaryotes, supports the movement of hundreds of distinct cargos, each with specific trafficking requirements. To achieve this functional diversity, dynein must bind to the multi-subunit complex dynactin and one of a family of cargo adaptors to be converted into an active, processive motor complex. Very little is known about the dynamic processes that promote the formation of this complex. To delineate the kinetic steps that lead to dynein activation, we developed a single-molecule fluorescence assay to visualize the real-time formation of dynein-dynactin-adaptor complexes in vitro. We found that dynactin and adaptors bind dynein independently rather than cooperatively. We also found that different dynein adaptors promote dynein-dynactin-adaptor assembly with dramatically different kinetics, which results in complex formation occurring via different assembly pathways. Despite differences in association rates or mechanism of assembly, all adaptors tested can generate a population of tripartite complexes that are very stable. Our work provides a model for how modulating the kinetics of dynein-dynactin-adaptor binding can be harnessed to promote differential dynein activation and reveals a new facet of the functional diversity of the dynein motor.

biophysics↗

bioRxiv2023

HIV-1 binds dynein directly to hijack microtubule transport machinery

Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dyneins tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically hijacks the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm.

microbiology↗

bioRxiv2023

Ndel1 modulates dynein activation in two distinct ways

Dynein is the primary minus-end-directed microtubule motor [1]. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex" [2, 3]. The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and adaptor [4, 5]. Ndel1 and its orthologue Nde1 are dynein and Lis1 binding proteins that help control where dynein localizes within the cell [6]. Cell-based assays suggest that Ndel1/Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear [6]. Using purified proteins and quantitative binding assays, we found that Ndel1s C-terminal region contributes to binding to dynein and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in Ndel1s C-terminal domain increase its ability to inhibit dynein-dynactin-adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.

biochemistry↗

bioRxiv2022

Lis1 binding regulates force-induced detachment of cytoplasmic dynein from microtubules

Cytoplasmic dynein-1 (dynein) is an AAA+ motor that transports intracellular cargos towards the microtubule minus end. Lissencephaly-1 (Lis1) binds to the AAA+ ring and stalk of dyneins motor domain and promotes the assembly of active dynein complexes. Recent studies showed that Lis1 slows motility when it remains bound to dynein, but the underlying mechanism remained unclear. Using single-molecule and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 does not slow dynein motility by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect the forces that stall dynein movement, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1 binding sites on dyneins stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1s interaction with the AAA+ ring is sufficient to result in slower movement and that Lis1s interaction with dyneins stalk slows force-induced detachment of dynein from microtubules.

biophysics↗

bioRxiv2022

The KASH5 protein involved in meiotic chromosomal movements is a novel dynein activating adaptor

Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.

cell biology↗

bioRxiv2021

Structural Basis for Cytoplasmic Dynein-1 Regulation by Lis1

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator that binds directly to dyneins motor domain, and by doing so alters dyneins mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1[A] structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1s {beta}-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1s ability to form fully active human dynein complexes and to regulate yeast dyneins mechanochemistry and in vivo function. We present a model for the conserved role of Lis1 in regulating dynein from yeast to humans.

cell biology↗

bioRxiv2019

Lis1 promotes the formation of maximally activated cytoplasmic dynein-1 complexes

Cytoplasmic dynein-1 is a molecular motor that drives nearly all minus-end-directed microtubule-based transport in human cells, performing functions ranging from retrograde axonal transport to mitotic spindle assembly1,2. Activated dynein complexes consist of one or two dynein dimers, the dynactin complex, and an \"activating adaptor\", with maximal velocity seen with two dimers present (Fig. 1a)3-6. Little is known about how this massive [~]4MDa complex is assembled. Using purified recombinant human proteins, we uncovered a novel role for the dynein-binding protein, Lis1, in the formation of fully activated dynein complexes containing two dynein dimers. Lis1 is required for maximal velocity of complexes activated by proteins representing three different families of activating adaptors: BicD2, Hook3, and Ninl. Once activated dynein complexes have formed, they do not require the presence of Lis1 for sustained maximal velocity. Using cryo-electron microscopy we show that human Lis1 binds to dynein at two sites on dyneins motor domain, similar to yeast dynein7. We propose that the ability of Lis1 to bind at these sites may function in multiple stages of assembling the motile human dynein/ dynactin/ activating adaptor complex.\n\nO_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC=\"FIGDIR/small/683052v1_fig1.gif\" ALT=\"Figure 1\">\nView larger version (37K):\norg.highwire.dtl.DTLVardef@1acc26corg.highwire.dtl.DTLVardef@1107393org.highwire.dtl.DTLVardef@113eab4org.highwire.dtl.DTLVardef@95f6f4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Lis1 increases microtubule binding and the velocity of activated dynein complexes. a. Schematic of the current model for dynein activation. Dynein is autoinihibited in the Phi conformation, opens and then adopts a parallel conformation that is seen in the activated dynein complex, which includes dynactin and an activating adaptor. Maximally activated dynein contains two dynein dimers (dynein A and B, far right) b. Schematic of the dynein motor domain with AAA+ ATPase domains colored in rainbow, highlighting the two Lis1 binding sites in yeast dynein, \"sitering\" at AAA3/4 and \"sitestalk\" on dyneins stalk, which leads to dyneins microtubule binding domain (MTBD). Lis1 is shown in orange to the right c. Schematic of the activating adaptor constructs used in this study. d. Binding density (mean {+/-} s.e.m. dynein) of dynein alone on microtubules in the absence (white circles) or presence (black circles) of 300 nM Lis1. Data was normalized to a density of 1.0 in the absence of Lis1. Statistical analysis was performed using a two-tailed unpaired t test; ****, p<0.0001; n = 12 replicates for each condition. e. Binding density (mean {+/-} s.e.m) of dynein/ dynactin/ activating adaptor complexes on microtubules in the absence (white circles) or presence (black circles) of 300 nM Lis1. The activating adaptors used are indicated. Data was normalized to a density of 1.0 in the absence of Lis1. Statistical analysis was performed using a two-tailed unpaired t test; ****, p<0.0001; n = 12 replicates for each condition. f. Velocity of dynein/ dynactin/ activating adaptor complexes in the absence (white circles) or presence (black circles) of 300 nM Lis1. The median and interquartile range are shown and the activating adaptors used are indicated. Statistical analysis was performed using a two-tailed Mann-Whitney test; ****, p<0.0001; ns, p=0.3498; n (individual single molecule events) = 506 (BicD2-S no Lis1), 569 (BicD2-S with Lis1), 496 (BicD2-L no Lis1), 505 (BicD2-L with Lis1), 454 (Hook3 no Lis1), 471 (Hook3 with Lis1), 490 (Ninl no Lis1), 582 (Ninl with Lis1). g. Percent processive runs (mean {+/-} s.e.m.) of dynein/ dynactin/ Hook3 complexes in a higher salt buffer (60 mM KOAc versus 30 mM KOAc in our standard motility buffer) in the absence (white circles) or presence (black circles) of 300 nM Lis1. Statistical analysis was performed using a one-way ANOVA and Tukeys multiple comparisons test; ****, p<0.0001; n = 3 replicates per condition. h. Schematic of the peroxisome relocation assay. i. Peroxisome velocity in human U2OS cells with scrambled or Lis1 siRNA knockdown. The median and interquartile range are shown. Statistical analysis was performed using a two-tailed unpaired t test; ***, p=0.0002; n (average peroxisome velocity per cell) = 30 per condition. More than 7 events were measured per cell.\n\nC_FIG

biochemistry↗