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eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
John P Gillies ◽  
Janice M Reimer ◽  
Eva P Karasmanis ◽  
Indrajit Lahiri ◽  
Zaw Min Htet ◽  
...  

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 known to bind directly to dynein's motor domain, and by doing so alters dynein's 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 3.1Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.


2022 ◽  
Author(s):  
Robert Fagiewicz ◽  
Corinne Crucifix ◽  
Celia Deville ◽  
Bruno Kieffer ◽  
Yves Nomine ◽  
...  

The cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is one of the most prominent examples within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis as well as interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2 and cytoplasmic dynein-1. We also describe pH-dependent conformational changes of BicD2 using cryoEM, template-free structure predictions, and biophysical tools. Our results will help defining the biochemical parameters for the invitro reconstitution of higher order BicD2 protein complexes.


2021 ◽  
Author(s):  
Wen Lu ◽  
Margot Lakonishok ◽  
Anna S. Serpinskaya ◽  
Vladimir I Gelfand

Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. We have previously shown that cytoplasmic dynein is required for Drosophila oocyte growth, and assumed that it transports cargoes along microtubule tracks from nurse cells to the oocyte. Here we report that instead transporting cargoes along microtubules into the oocyte, cortical dynein actively moves microtubules in nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. We demonstrate this microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein can perform bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. We propose that the dynein-driven microtubule flow could serve as a novel mode of cargo transport for fast cytoplasmic transfer to support rapid oocyte growth.  


2021 ◽  
Author(s):  
Joshua W Thompson ◽  
Maria F. Valdes Michel ◽  
Bryan T Phillips

The C. elegans Wnt/β-catenin Asymmetry (WβA) pathway utilizes asymmetric regulation of SYS-1/β-catenin and POP-1/TCF coactivators. This differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate at birth. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific mobility of centrosomal SYS-1 and 'traffic jam' like enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe that depletion of microtubules by Nocodazole treatment or RNAi of putative dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose retrograde microtubule-mediated trafficking enables SYS-1 and negative regulators to enrich at centrosomes, enhancing their interaction and perhaps implicating the centrosome as a mitotic sink for proteins targeted for degradation.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Spencer D. Balay ◽  
Tobias Hochstoeger ◽  
Alexandra Vilceanu ◽  
E. Pascal Malkemper ◽  
William Snider ◽  
...  

AbstractCryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton.


2021 ◽  
Author(s):  
Shahar Taiber ◽  
Oren Gozlan ◽  
Roie Cohen ◽  
Leonarde Andrade ◽  
Yehu Moran ◽  
...  

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, nesprin-4 (encoded by Syne4) was shown to play a crucial role in nucleus positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in-vivo AAV gene delivery, we show that this interaction is critical for nucleus positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Overall, our results suggest that OHCs require unique cellular machinery for proper nucleus positioning at the onset of electromotility. This machinery relies on the interaction between nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nucleus positioning.


2021 ◽  
pp. mbc.E21-06-0295
Author(s):  
Carlos M. Guardia ◽  
Akansha Jain ◽  
Rafael Mattera ◽  
Alex Friefeld ◽  
Yan Li ◽  
...  

Autophagy-related protein 9 (ATG9) is a transmembrane protein component of the autophagy machinery that cycles between the trans-Golgi network (TGN) in the perinuclear area and other compartments in the peripheral area of the cell. In mammalian cells, export of the ATG9A isoform from the TGN into ATG9A-containing vesicles is mediated by the adaptor protein 4 (AP-4) complex. However, the mechanisms responsible for the subsequent distribution of these vesicles to the cell periphery is unclear. Herein we show that the AP-4–accessory protein RUSC2 couples ATG9A-containing vesicles to the plus-end-directed microtubule motor kinesin-1 via an interaction between a disordered region of RUSC2 and the kinesin-1 light chain (KLC). This interaction is counteracted by the microtubule-associated WD40-repeat domain 47 protein (WDR47). These findings uncover a mechanism for the peripheral distribution of ATG9A-containing vesicles, involving the function of RUSC2 as a kinesin-1 adaptor and WDR47 as a negative regulator of this function.


2021 ◽  
Author(s):  
Chiara Cassioli ◽  
Anna Onnis ◽  
Francesca Finetti ◽  
Nagaja Capitani ◽  
Jlenia Brunetti ◽  
...  

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here we have investigated the potential role of BBS1, an essential core component of the Bardet-Biedl syndrome complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrate that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH, a process that we demonstrate to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that acts upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anja Beckers ◽  
Franziska Fuhl ◽  
Tim Ott ◽  
Karsten Boldt ◽  
Magdalena Maria Brislinger ◽  
...  

AbstractCilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.


2021 ◽  
Author(s):  
John P Gillies ◽  
Janice M Reimer ◽  
Eva P Karasmanis ◽  
Indrajit Lahiri ◽  
Zaw Min Htet ◽  
...  

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 dynein's motor domain, and by doing so alters dynein's 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 3.1Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's β-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function. We present a model for the conserved role of Lis1 in regulating dynein from yeast to humans.


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