Biology subjects

del Castillo, U.

Publications and source records attributed to del Castillo, U..

5 recordsLinked to original sources

bioRxiv2026

AR-V7 Utilizes a Noncanonical Nuclear Localization Signal to Sustain Androgen-Independent Nuclear Import and Signaling

AR-V7 is the most prevalent androgen receptor splice variant in metastatic castration-resistant prostate cancer, yet how it enters the nucleus independently of androgen binding has remained unclear. We previously demonstrated that AR-V7 undergoes nuclear import via a non-canonical pathway distinct from the microtubule-based, importin-/{beta}- and Ran-dependent mechanism used by full-length AR. Here, using systematic truncation and alanine-scanning mutagenesis, we identify a non-classical nuclear localization motif at the junction of the DNA-binding domain (DBD) and cryptic exon 3 (CE3) that is required for efficient AR-V7 nuclear import. Specific basic residues within the DBD and CE3 are required not only for nuclear entry but also for transcriptional activity. Unexpectedly, mutants retaining partial nuclear localization were transcriptionally silent, suggesting that CE3 contributes to transcriptional engagement through mechanisms separate from nuclear transport. Genome-wide transcriptomic analysis of a strongly import-deficient mutant confirmed that disruption of nuclear entry abolishes the AR-V7 transcriptional program. To map compartment-specific protein interactions, we applied antibody-guided photocatalytic proximity labeling. Nuclear AR-V7 associated most prominently with both canonical BAF and PBAF SWI/SNF chromatin remodeling complexes, together with known transcriptional coregulators and DNA-repair factors, whereas the cytoplasm-restricted mutant engaged different networks centered on mRNA deadenylation and decay, cytoskeletal regulation, and chaperone systems. Together, these findings define a variant-specific NLS, separate nuclear import from CE3-dependent transcriptional competence, and identify the cytoplasmic networks that become accessible when AR-V7 nuclear entry is impaired. They reveal two mechanistically distinct vulnerabilities for suppressing AR-V7 signaling: blocking nuclear translocation and disrupting CE3-dependent transcriptional engagement within the nucleus.

cancer biology↗

bioRxiv2026

CAR-T Targeting of Mutant Calreticulin Establishes a Potentially Curative Stem Cell-Directed Therapy for Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are sustained by mutated hematopoietic stem cells (HSCs). Existing therapies fail to eliminate this compartment, leaving allogeneic HSC transplantation as the only curative option. Recurrent MPN driver mutations in calreticulin (CALRmut) generate a C-terminal neopeptide that requires cell-surface expression for oncogenic signaling, making it an attractive immunologic target. However, it remains unknown if CALRmut is uniformly displayed on all MPN HSCs within hematopoietic microenvironments. We generated huAB2, a high-affinity CALRmut-specific humanized antibody, to use as the targeting domain for chimeric antigen receptor (CAR)-T cells. We show that CALRmut is consistently displayed on functional MPN HSCs and accessible in vivo. huAB2 CAR-T cells eradicate MPN-propagating CALRmut HSCs in patient-derived tumor xenograft models without antigen escape while preserving coexisting normal human and host hematopoiesis. These findings establish CALRmut display as an obligate feature of MPN HSC fitness and support the feasibility of curative, non-transplant immunotherapy for CALRmut MPNs. SignificanceTherapies that eradicate cancer stem cells enable cure, but their feasibility is unknown. We establish an approach to potentially cure MPNs by proving mutant calreticulin to be a MPN stem cell marker that can be targeted by CAR-T cells to selectively wipe out disease in preclinical models of human MPNs.

cancer biology↗

bioRxiv2021

Ataxin-2 is essential for neurodevelopment in Drosophila and is a major regulator of the cytoskeleton.

Ataxin-2 (Atx2) is a highly conserved RNA binding protein. Atx2 undergoes polyglutamine expansion leading to Amyotrophic Lateral Sclerosis (ALS) or Spinocerebellar Ataxia type 2 (SCA2). However, the physiological functions of Atx2 in neurons remain unknown. Here, using the powerful genetics of Drosophila, we show that Atx2 is essential for normal neuronal cytoskeletal dynamics and organelle trafficking. Upon neuron-specific Atx2 loss, the microtubule and actin networks were abnormally stabilized and cargo transport was drastically inhibited. Depletion of Atx2 caused multiple morphological defects in the nervous system of 3rd instar larvae. These include reduced brain size, impaired optic lobe innervation and decreased dendrite outgrowth. Defects in the nervous system caused loss of the ability to crawl and lethality at the pupal stage. Taken together, these data mark Atx2 as a major regulator of cytoskeletal dynamics and denote Atx2 as an essential gene in neurodevelopment, as well as a neurodegenerative factor.

cell biology↗

bioRxiv2020

Kinetochore protein Spindly controls microtubule polarity in Drosophila axons

Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules. Significance StatementNeurons send and receive electrical signals through long microtubule-filled neurites called axons and dendrites. One of the main structural differences between axons and dendrites is how their microtubules are organized. Axons contains microtubules with their plus-ends out while microtubules in dendrites are organized with mixed or plus-end-in orientation. Dynein, the main minus-end microtubule motor, anchored to cortical actin filaments in the axons is responsible for the uniform microtubule polarity in axons. However, it is unknown how dynein is recruited to the actin cortex in axons. The major finding of this work is that Spindly, a protein involved in anchoring dynein to kinetochores during cell division, has a second important function in interphase cells recruiting dynein to the actin cortex in axons.

cell biology↗

bioRxiv2019

Ser/Thr kinase Trc controls neurite outgrowth in Drosophila by modulating microtubule-microtubule sliding

Correct neuronal development requires tailored neurite outgrowth. Neurite outgrowth is driven by microtubule sliding - the transport of microtubules along each other. We have recently demonstrated that a \"mitotic\" kinesin-6 (Pavarotti in Drosophila) effectively inhibits microtubule-sliding and neurite outgrowth. However, mechanisms of Pavarotti regulation in interphase cells and specifically in neurite outgrowth are unknown. Here, we use a combination of live imaging and biochemical methods to show that the inhibition of microtubule sliding by Pavarotti is controlled by phosphorylation. We identify the Ser/Thr NDR kinase Tricornered (Trc) as a Pavarotti-dependent regulator of microtubule sliding in neurons. Further, we show that Trc-mediated phosphorylation of Pavarotti promotes its interaction with 14-3-3 proteins. 14-3-3 binding is necessary for Pavarotti to interact with microtubules and inhibit sliding. Thus, we propose a pathway by which microtubule sliding can be up or down regulated in neurons to control neurite outgrowth, and establish parallels between microtubule sliding in mitosis and post-mitotic neurons.

cell biology↗