Biology subjects

Neiswender, H.

Publications and source records attributed to Neiswender, H..

9 recordsLinked to original sources

bioRxiv2026

An in vivo examination of Dynein-Cargo complex formation.

Long-range intracellular transport relies on microtubule motors. This process is particularly important in large cells such as neurons and oocytes. While transport towards the plus-end of microtubules utilizes many kinesins, minus-end transport is largely mediated by a single motor, cytoplasmic dynein. Activation of dynein requires the large dynactin complex as well as a cargo adaptor. How dynein, dynactin, and adaptors assemble in vivo, particularly within specialized tissues such as the Drosophila egg chamber remains unclear. In the current study, we defined the dynein interactome in Drosophila egg chambers using in vivo proximity biotin ligation. Our findings suggest that Bicaudal-D (BicD) is the principal adaptor responsible for activating dynein and linking it with cargo in this tissue. We also identified Centrocortin (Cen) as a dynein adaptor in egg chambers. However, unlike BicD, loss of Cen did not affect dynein localization or apparent activation. To more specifically analyze adaptor-dependent assembly and cargo transport, we examined dynein light intermediate chain (Dlic) mutants known to impair adaptor binding. As expected, these mutants disrupted the BicD-dynein interaction. However, Cen remained associated with the dynein/dynactin complex in the mutant background, suggesting that Cen engages the motor by a different mechanism. Finally, live imaging of microtubules revealed that even when adaptor binding is compromised, dynein-driven microtubule gliding can still deliver nurse cell-derived cargo into the oocyte, albeit with reduced efficiency. Collectively, our results reveal multiple, mechanistically distinct routes for adaptor association with dynein in vivo and indicate that redundant processes can sustain cargo transport during oogenesis.

cell biology↗

bioRxiv2026

Proteomic profiling of cytoskeletal interactomes using MT-ID and Act-ID.

The microtubule and actin cytoskeletons form dynamic, interconnected networks that are critical for eukaryotic cell function. These networks govern intracellular organization, cargo transport, cell migration, and tissue morphogenesis. Microtubules and actin filaments are regulated by diverse binding proteins that control many aspects of their function. However, identifying cytoskeletal-interacting proteins has been challenging due to the transient and weak nature of many interactions and the disruption of native architecture by conventional biochemical approaches. These limitations suggest that numerous physiologically relevant cytoskeletal regulators remain undiscovered. Identifying these factors requires novel and sensitive methodologies that can capture cytoskeletal interactions under native cellular conditions. Here, we present MT-ID and Act-ID, powerful proximity-labeling tools for identifying microtubule and actin-interacting proteins, respectively. MT-ID employs the microtubule-binding domain of MAP7 (EMTB) fused to TurboID, a highly active promiscuous biotin ligase. Act-ID utilizes the actin-binding domain of ITPKA (F-tractin) similarly fused to TurboID. We validate both approaches by successfully identifying numerous known cytoskeletal regulators and discovering potentially novel interacting proteins. Functional characterization reveals that LIMCH1 is a previously unrecognized microtubule-associated protein whose depletion increases microtubule density. Additionally, we identify FBXO30 as a novel actin-interacting protein, with its loss promoting increased focal adhesion formation. MT-ID and Act-ID will be useful not only to identify cytoskeletal interacting proteins but also to define changes to the cytoskeletal interactome when cells are exposed to changing physiological conditions.

cell biology↗

bioRxiv2026

To Be or Not to Be an Oocyte: Msps/XMAP215 Controls Oocyte Cell Fate in the Drosophila Ovary

Cell fate determination, the process by which cells commit to specific identities, is fundamental to development. Oocyte specification in Drosophila provides an excellent model to dissect these molecular mechanisms. In the Drosophila ovary, 16 interconnected germline cells arise through four mitotic divisions with incomplete cytokinesis. Among them, the two oldest cells become pro-oocytes, and one is selected to develop into the oocyte while maintaining diploidy; the remaining 15 differentiate into nurse cells and enter endoreplication. Previously, we showed that Mini spindles (Msps), a microtubule polymerase and homolog of XMAP215, is essential for oocyte maintenance. Here, we report that Msps/XMAP215 is expressed early in development, with both mRNA and protein enriched in the pro-oocytes prior to oocyte specification. Knockdown of Msps prevents oocyte specification, leading to egg chambers with 16 nurse cells. Loss of Msps also disrupts the accumulation of the oocyte marker Orb and the microtubule minus-end binding protein Patronin/CAMSAP, both critical for oocyte specification. Remarkably, optogenetic recruitment of Msps is sufficient to increase microtubule polymerization and promote Orb accumulation in nurse cells, suggesting that Msps activity is sufficient to drive oocyte fate determination. Furthermore, we show that Msps associates with the germline-specific organelles, spectrosome and fusome, and becomes asymmetrically distributed among sister cells, allowing the two pro-oocytes to inherit higher levels of Msps than their siblings. Together, we propose a model in which Msps-mediated microtubule polymerization provides the pro-oocyte with a competitive advantage, initiating a positive feedback loop that involves dynein-dependent transport of msps mRNA to reinforce oocyte specification. TeaserA self-reinforcing cytoskeletal feedback loop selects a single egg cell from a cluster of interconnected germ cells.

cell biology↗

bioRxiv2025

Dominant spinal muscular atrophy linked mutations in the cargo binding domain of BICD2 result in altered interactomes and dynein hyperactivity

Cytoplasmic dynein-1 (dynein) is responsible for the transport of most cellular cargo towards the minus end of microtubules. Dynein activation requires the multi-subunit dynactin complex and an activating cargo adaptor. The adaptors serve to link dynein with cargo and to fully activate the motor. Mutations in one of these activating adaptors, Bicaudal-D2 (BICD2), are associated with a neurodegenerative disease called Spinal Muscular Atrophy with Lower Extremity Predominance (SMALED2). The molecular defect that underlies SMALED2 is largely unknown. In addition to interacting with dynein, BICD2 has also been shown to associate with KIF5B, a plus-end directed microtubule motor. We hypothesized that interactome changes associated with mutant versions of BICD2, and the resulting differences in cargo transport, might underlie the etiology of SMALED2. To test our hypothesis, we first defined the interactome of wild-type BICD2. This led to the identification of known BICD2 interacting proteins in addition to potentially novel cargo such as components of the HOPS complex, a six-subunit complex involved in endo-lysosomal trafficking. We next determined the interactome of three SMALED2 linked mutants in BICD2, two of which reside in the cargo binding domain. Interestingly, all three mutations resulted in BICD2-mediated dynein hyper-activation. Furthermore, all three mutants were associated with interactome changes. One of these mutants, BICD2_R747C, was deficient in binding to HOPS complex components and the nucleoporin RANBP2. In addition, this mutant also resulted in a gain of function interaction with GRAMD1A, a protein localized to the endoplasmic reticulum. This gain of function interaction resulted in mis-localization of GRAMD1A in BICD2_R747C expressing cells. Collectively, our results suggest that dynein hyperactivity, interactome changes, and cargo transport defects might contribute to the symptoms associated with SMALED2.

cell biology↗

bioRxiv2023

The Bicaudal-D/Egalitarian complex defines the specificity of cargo transport by Dynein.

Numerous motors of the Kinesin family contribute to plus-end microtubule transport. However, almost all transport towards the minus-end of microtubules involves a single motor, cytoplasmic Dynein (Dynein). To gain motility, Dynein must interact with activating cargo adaptors. One such adaptor is Bicaudal-D (BicD; BICD2 in humans). Mutations in BICD2 are associated with Spinal Muscular Atrophy (SMA), a degenerative motor neuron disease. BicD is autoinhibited from binding Dynein in the absence of cargo. A well-characterized cargo for BicD is the RNA binding protein, Egalitarian (Egl). Egl in conjunction with BicD links mRNA to Dynein in the Drosophila egg chamber and embryo. To better understand how Dynein is activated and whether BicD links additional cargo with Dynein, we defined the BicD interactome in the presence and absence of Egl. This revealed a vast number of potentially novel BicD cargos including the nucleoporin Nup358/RANBP2, a known cargo of mammalian BICD2. In strains depleted of Egl, BicD remained associated with most of its cargo including Nup358. However, the interaction of BicD with Dynein was reduced. Consequently, the localization of Nup358 and its association with Dynein was disrupted. Thus, while BicD can bind diverse cargos, linking these cargos with Dynein requires Egl. Furthermore, our studies revealed that a SMA associated mutation in the cargo binding domain of BicD enhanced the Dynein mediated localization of certain cargos but disrupted the localization of others. At the organismal level, this mutation resulted in compromised mobility. Specific transport defects might therefore underlie the etiology of BicD associated SMA.

cell biology↗

bioRxiv2021

In vivo proximity biotin ligation identifies the interactome of Egalitarian, a Dynein cargo adaptor.

Numerous motors of the Kinesin family contribute to plus-end directed microtubule transport. However, almost all transport towards the minus-end of microtubules involves Dynein. Understanding the mechanism by which Dynein transports this vast diversity of cargo is the focus of intense research. In select cases, adaptors that link a particular cargo with Dynein have been identified. However, the sheer diversity of cargo suggests that additional adaptors must exist. We used the Drosophila egg chamber as a model to address this issue. Within egg chambers, Egalitarian is required for linking mRNA with Dynein. However, in the absence of Egalitarian, Dynein transport into the oocyte is severely compromised. This suggests that additional cargoes might be liked to Dynein in an Egalitarian-dependent manner. We therefore used proximity biotin ligation to define the interactome of Egalitarian. This approach yielded several novel interacting partners, including P body components and proteins that associate with Dynein in mammalian cells. We also devised and validated a nanobody based proximity biotinylation strategy which can be used to define the interactome of any GFP-tagged protein.

cell biology↗

bioRxiv2021

Dynein light chain-dependent dimerization of Egalitarian is essential for maintaining oocyte fate in Drosophila.

Egalitarian (Egl) is an RNA adaptor for the Dynein motor and is thought to link numerous, perhaps hundreds, of mRNAs with Dynein. Dynein, in turn, is responsible for the transport and localization of these mRNAs. Studies have shown that efficient mRNA binding by Egl requires the protein to dimerize. We recently demonstrated that Dynein light chain (Dlc) is responsible for facilitating the dimerization of Egl. Mutations in Egl that fail to interact with Dlc do not dimerize, and as such, are defective for mRNA binding. Consequently, this mutant does not efficiently associate with BicaudalD (BicD), the factor responsible for linking the Egl/mRNA complex with Dynein. In this report, we tested whether artificially dimerizing this Dlc-binding mutant using a leucine zipper would restore mRNA binding and rescue mutant phenotypes in vivo. Interestingly, we found that although artificial dimerization of Egl restored BicD binding, it only partially restored mRNA binding. As a result, Egl-dependent phenotypes, such as oocyte specification and mRNA localization, were only partially rescued. We hypothesize that Dlc-mediated dimerization of Egl results in a three-dimensional conformation of the Egl dimer that is best suited for mRNA binding. Although the leucine zipper restores Egl dimerization, it likely does not enable Egl to assemble into the conformation required for maximal mRNA binding activity.

cell biology↗

bioRxiv2020

Acute knockdown of extracellular matrix protein Tinagl1 disrupts heart laterality and pronephric cilia in zebrafish embryonic development

A highly-conserved extracellular matrix protein, Tinagl1, modulates Wnt, integrin, TGF-{beta}, and EGF-R signaling in vitro, but its significance in vivo has remained in doubt. To bypass possible genetic compensation by an ortholog encoded exclusively in mammalian genomes, we examine Tinagl1 function in zebrafish embryos. In this model, tinagl1 mRNA is detected in the developing spinal cord and pronephros. Acute knockdown using either CRISPR/Cas9 somatic mutagenesis or splice-blocking morpholinos reveals left-right (LR) heart looping defects, pronephros dilatations, and ventral body curvature. This constellation of defects characteristically results from the loss of motile cilia function, and we confirm the presence of shortened and fewer cilia in the pronephric duct and in the Kupffers vesicle where LR asymmetry is established. A link to known Wnt3a/{beta}-catenin signaling that activates the motile cilia transcriptional program is supported by manipulation of Wnt3a and {beta}-catenin levels in tinagl1 knockdown embryos. In addition to ciliopathy-like defects, the tinagl1 knockdown shows disorganization of longitudinal axon tracts in the spinal cord and defects in motor neuron outgrowth. Together, these results provide evidence that Tinagl1 is important in development, and that zebrafish is an ideal model in which to explore its relationships to cilia and secreted signaling molecules.

developmental biology↗

bioRxiv2019

Egalitarian binding partners, Dynein Light Chain and Bicaudal-D, act sequentially to link mRNA to the Dynein motor

A widely conserved mechanism of polarity establishment is the localization of mRNA to specific cellular regions. While it is clear that many mRNAs are transported to their destinations along microtubule tracks, much less is known regarding the mechanism by which these mRNAs are linked to microtubule motors. The RNA binding protein Egalitarian (Egl) is necessary for localization of several mRNAs in Drosophila oocytes and embryos. In addition to binding RNA, Egl also interacts with Dynein light chain (Dlc) and Bicaudal-D (BicD). The role of Dlc and BicD in mRNA localization has remained elusive. Like Egl, both proteins are required for oocyte specification. Null alleles in these genes result in an oogenesis block. In this report, we used an innovative approach to overcome the oogenesis block. Our findings reveal that the primary function of Dlc is to promote Egl dimerization. Loss of dimerization compromises the ability of Egl to bind RNA. Consequently, Egl is not bound to cargo, and is not able to efficiently associate with BicD and the Dynein motor. Our results therefore identify the key molecular steps required for assembling a localization competent mRNP.

cell biology↗