Biology subjects

Prekeris, R.

Publications and source records attributed to Prekeris, R..

17 recordsLinked to original sources

bioRxiv2026

NET1 mRNA localization to the midbody is required for ARP2/3-dependent initiation of mitotic abscission

The recruitment and activation of abscission machinery following mitosis is tightly regulated in time and space, yet the mechanisms controlling this process are poorly understood. We find that RNA localization and local translation at the midbody regulates when and where abscission-regulating proteins are expressed. The 3'UTR of NET1 mRNA contains an element that is necessary and sufficient for RNA targeting to the midbody. Mislocalization of NET1 mRNA results in a loss of NET1 protein, a Rho family GEF, throughout the intercellular bridge as well as slower cell proliferation and delayed abscission. This leads to a loss of Arp2/3 at the abscission site and is dependent upon NET1 binding to Rho family GTPases. These findings establish midbody RNA localization and local translation as a key layer of regulation over abscission timing and identify a role for NET1 as a regulator of Arp2/3-mediated branched actin accumulation at the abscission site.

molecular biology↗

bioRxiv2025

TPGS1 Regulates Central Spindle Microtubule Glutamylation and Remodeling During Telophase and Abscission

Microtubules perform a variety of cellular functions including regulation of mitotic cell division, cilia formation, and neurite extension. Post-translational modifications controlled by the TTLL-family of enzymes confer a host of properties that affect microtubule dynamics and function. Specifically, polyglutamylation of tubulin C-terminal tails plays an important role in regulating microtubule dynamics and function within specific cellular contexts. In this paper we examined contributions from and potential regulators of polyglutamylation during mitosis, focusing on the microtubule remodeling that occurs in telophase once the mitotic spindle has completed chromosome separation. We demonstrate that the anaphase-to-telophase transition is accompanied by an increase in short-chain polyglutamylation of central spindle microtubules. We also show that TTLL1 and TPGS1, subunits of the tubulin polyglutamylation complex, are targeted to the intracellular bridge and midbody during cell progression through telophase. Finally, we demonstrate that loss of TPGS1 leads to defects in remodeling of the central spindle during telophase and impacts the cells ability to complete mitotic cell division.

cell biology↗

bioRxiv2025

Mono-Ubiquitylation-Dependent Rap2 Activation Regulates Lamellipodia Dynamics During Cell Migration

Cell migration is a complex process hallmarked by front-to-back cell polarity that is established by the highly dynamic actin cytoskeleton. Branched actin polymerization creates a lamellipodia at the leading edge of the cell, while the contractile acto-myosin cytoskeleton is present at the lagging edge. Rap2, a Ras GTPase family member, has previously been reported to localize to the lamellipodia as a result of Rab40/CRL5 E3 ubiquitin ligase induced tri-mono-ubiquitylation. However, how Rap2 functions and how mono-ubiquitylation targets Rap2 to the lamellipodia remained unclear. Here, we demonstrate that Rap2 is recruited to retracting lamellipodia ruffles where it inhibits RhoA and regulates lamellipodia dynamics and facilitates cell migration. Furthermore, using a variety of genetic and pharmacological techniques, we show that tri-mono-ubiquitylation is required for GEF-dependent Rap2 activation, a necessary step for Rap2 targeting to lamellipodia membrane. As such, we demonstrate how this unique mono-ubiquitylation of Rap2 regulates lamellipodia actin dynamics during cell migration. SUMMARYMono-ubiquitylation is necessary for activity and localization of the Rap2 GTPase. Here, we report that Rap2 lamellipodia and membrane dwell time is facilitated by mono-ubiquitylation dependent GEF activation. Furthermore, we demonstrate that mono-ubiquitylated Rap2 regulates lamellipodia dynamics through inhibiting RhoA activity.

cell biology↗

bioRxiv2024

Rab40 GTPases regulate AMBRA1-mediated transcription and cell migration

The Rab40 subfamily are unique small monomeric GTPases that form CRL5-based ubiquitin E3 ligase complex and regulate ubiquitylation of specific target proteins. Recent studies have shown that Rab40s play an important role in regulating cell migration, but the underlying mechanisms of Rab40/CRL5 complex function are still not fully understood. In this study we identified AMBRA1 as a novel binding partner of Rab40 GTPases and showed that this interaction mediates a bi-directional crosstalk between CRL4 and CRL5 E3 ligases. Importantly, we found that Rab40/CRL5 ubiquitylates AMBRA1, which does not result in AMBRA1 degradation, but instead it seems to induce AMBRA1-dependent regulation of gene transcription. The global transcriptional profiles identified by RNA-seq showed that AMBRA1 regulates transcription of genes related to cell adhesion and migration. Additionally, we have shown that AMBRA1-dependent transcription regulation does not require the enzymatic activity of AMBRA1/CRL4, and that Rab40-induced AMBRA1 ubiquitylation leads to dissociation of AMBRA1/CRL4 complex. Taken together, our findings reveal a novel function of Rab40/CRL5 complex as an important regulator for AMBRA1-dependent transcription of genes involved in cell migration.

molecular biology↗

bioRxiv2023

TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the /{beta}-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

cell biology↗

bioRxiv2023

HOPS-dependent lysosomal fusion controls Rab19 availability for ciliogenesis in polarized epithelial cells

Primary cilia are sensory cellular organelles crucial for organ development and homeostasis. Ciliogenesis in polarized epithelial cells requires Rab19-mediated clearing of apical cortical actin to allow the cilium to grow from the apically-docked basal body into the extracellular space. Loss of the lysosomal membrane-tethering HOPS complex disrupts this actin-clearing and ciliogenesis, but it remains unclear how ciliary function of HOPS relates to its canonical function in regulating late endosome-lysosome fusion. Here, we show that disruption of HOPS-dependent lysosomal fusion indirectly impairs actin-clearing and ciliogenesis by disrupting the targeting of Rab19 to the basal body. We also find that Rab19 functions in endolysosomal cargo trafficking apart from its previously-identified role in ciliogenesis. In summary, we show that inhibition of lysosomal fusion abnormally accumulates Rab19 on late endosomes, thus depleting Rab19 from the basal body and thereby disrupting Rab19-mediated actin-clearing and ciliogenesis. Summary statementLoss of HOPS-mediated lysosomal fusion indirectly blocks apical actin clearing and ciliogenesis in polarized epithelia by trapping Rab19 on late endosomes and depleting Rab19 from the basal body.

cell biology↗

bioRxiv2022

The Novel Role of Midbody-Associated mRNAs in Regulating Abscission

Midbodies (MBs) have been shown to function during telophase as a recruiting hub, especially for ESCRT-III complex subunits, to regulate the abscission step of cytokinesis. However, the molecular machinery governing specific protein targeting and activation at the MB remains poorly understood. Until recently, it was thought that abscission regulating proteins, such as ESCRT-III complex subunits, accumulate at the MB by directly or indirectly binding to the MB resident protein, CEP55. However, recent studies have shown that depletion of CEP55 does not fully block ESCRT-III targeting to the MB, and cells in CEP55 knock-out mice divide normally. Additionally, since MBs are microtubule-rich, proteinaceous structures, it is conceptually hard to imagine how large protein complexes, such as the ESCRT-III complex, can successfully diffuse into the MB from the cytosol in a rapid and highly regulated manner. Here, we show that MBs contain mRNAs and that these MB-associated mRNAs can be locally translated, resulting in the accumulation of abscission-regulating proteins. We also demonstrate that localized MB-associated translation of CHMP4B is required for its targeting to the abscission site and that 3' UTR-dependent CHMP4B mRNA targeting to the MB is required for successful completion of cytokinesis. Finally, we identify regulatory cis-elements within RNAs that are necessary and sufficient for mRNA trafficking to the MB. Based on all this data, we propose a novel method of regulating cytokinesis and abscission by MB-associated targeting and localized translation of selective mRNAs.

cell biology↗

bioRxiv2022

cdon and boc affect trunk neural crest cell migration through a non-cell autonomous reduction of hedgehog signaling in zebrafish slow-twitch muscle

The transmembrane proteins cdon and boc are implicated in regulating hedgehog signaling during vertebrate development. Recent work showing roles for these genes in axon guidance and neural crest cell migration suggest that cdon/boc may play additional functions in regulating directed cell movements. We use novel and existing mutants to investigate a role for cdon and boc in zebrafish neural crest cell migration. We find that single mutant embryos exhibit normal neural crest phenotypes, but that neural crest migration is strikingly disrupted in double cdon;boc mutant embryos. We further show that this migration phenotype is associated with defects to the differentiation of slow-twitch muscle cells, and the loss of a Col1a1a containing extracellular matrix, suggesting that neural crest defects are a secondary consequence to defects in mesoderm development. Combined, our data add to a growing literature showing that cdon and boc act synergistically to promote hedgehog signaling during vertebrate development, and provide a foundation for using zebrafish to study the function of hedgehog receptor paralogs.

developmental biology↗

bioRxiv2021

Rab40c Regulates Focal Adhesions and Protein Phosphatase 6 Activity by Controlling ANKRD28 Ubiquitylation and Degradation

Rab40c is a SOCS box-containing protein which binds Cullin5 to form a ubiquitin E3 ligase complex (Rab40c/CRL5) to regulate protein ubiquitylation. However, the exact functions of Rab40c remain to be determined, and what proteins are the targets of Rab40c-Cullin5 mediated ubiquitylation in mammalian cells are unknown. Here we showed that in migrating MDA-MB-231 cells Rab40c regulates focal adhesions number, size, and distribution. Mechanistically, we found that Rab40c binds the protein phosphatase 6 (PP6) complex and ubiquitylates one of its subunits, ankyrin repeat domain 28 (ANKRD28), thus, leading to its lysosomal degradation. Furthermore, we identified that phosphorylation of FAK and MOB1 is decreased in Rab40c knock-out cells, which may contribute to focal adhesion site regulation by Rab40c. Thus, we propose a model where Rab40c/CRL5 regulates ANKRD28 ubiquitylation and degradation, leading to a decrease in PP6 activity, which ultimately affects FAK and Hippo pathway signaling to alter focal adhesion dynamics.

cell biology↗

bioRxiv2021

Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration

Cell migration is a complex process that involves coordinated changes in membrane transport, actin cytoskeleton dynamics, and extracellular matrix remodeling. Ras-like small monomeric GTPases, such as Rap2, play a key role in regulating actin cytoskeleton dynamics and cell adhesions. However, how Rap2 function, localization, and activation are regulated during cell migration is not fully understood. We previously identified the small GTPase Rab40b as a regulator of breast cancer cell migration. Rab40b contains a Suppressor of Cytokine Signaling (SOCS) box, which facilitates binding to Cullin5, a known E3 Ubiquitin Ligase component responsible for protein ubiquitylation. In this study, we show that the Rab40b/Cullin5 complex ubiquitylates Rap2. Importantly, we demonstrate that ubiquitylation regulates Rap2 activation, as well as recycling of Rap2 from the endolysosomal compartment to the lamellipodia of migrating breast cancer cells. Based on these data, we propose that Rab40b/Cullin5 ubiquitylates and regulates Rap2-dependent actin dynamics at the leading-edge, a process that is required for breast cancer cell migration and invasion. SUMMARYThe Rab40b/Cul5 complex is an emerging pro-migratory molecular machine. Duncan et al. identify the small GTPase Rap2 as a substrate of the Rab40b/Cul5 complex. They provide evidence that Rab40b/Cul5 ubiquitylates Rap2 to regulate its localization and activity during breast cancer cell migration, ultimately proposing a model by which Rap2 is targeted to the leading-edge plasma membrane to regulate actin dynamics during cell migration.

cell biology↗

bioRxiv2021

Trisomy 21 induces pericentrosomal crowding disrupting early stages of primary ciliogenesis and mouse cerebellar development

Primary cilia are signaling organelles essential for development and homeostasis. Loss of primary cilia is lethal, and decreased or defective cilia cause multisystemic conditions called ciliopathies. Down syndrome shares clinical overlap with ciliopathies. We previously showed that trisomy 21 diminishes primary cilia formation and function due to elevated Pericentrin, a centrosome protein encoded on chromosome 21. Pericentrin is mislocalized, creating aggregates that disrupt pericentrosomal trafficking and microtubule organization. Here, we examine the cilia-related molecules and pathways disrupted in trisomy 21 and their in vivo phenotypic relevance. Utilizing ciliogenesis time course experiments, we reveal how Pericentrin, microtubule networks, and components of ciliary vesicles are reorganized for ciliogenesis in euploid cells. Early in ciliogenesis, chromosome 21 polyploidy results in elevated Pericentrin and microtubule networks away from the centrosome that ensnare MyosinVA and EHD1, blocking mother centriole uncapping that is essential for ciliogenesis. Ciliated trisomy 21 cells have persistent trafficking defects that reduce transition zone protein localization, which is critical for Sonic hedgehog signaling. Sonic hedgehog signaling is decreased and anticorrelates with Pericentrin levels in trisomy 21 primary mouse embryonic fibroblasts. Finally, we observe decreased ciliation in vivo. A mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers. Our work reveals that elevated Pericentrin in trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding results in signaling defects consistent with the neurological deficits found in individuals with Down syndrome.

cell biology↗

bioRxiv2021

Trisomy 21 increases microtubules and disrupts centriolar satellite localization

Trisomy 21, the cause of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. Here we investigate the mechanisms by which PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT protein accumulates around the centrosome as a pericentrosomal cluster of enlarged cytoplasmic puncta that localize along and at MT ends. Cytoplasmic PCNT puncta impact the intracellular MT trafficking network required for primary cilia, as the PCNT puncta sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins, PCM1, CEP131 and CEP290, important for ciliogenesis, accumulate at sites of enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta, reestablish a normal density of MTs around the centrosome, restore ciliogenesis to wild type levels and decrease pericentrosomal crowding. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the microtubule network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia. TOCMcCurdy et al explore why elevated Pericentrin in trisomy 21 negatively impacts primary cilia formation and signaling. They find that elevated Pericentrin produces more pericentrosomal puncta that associates with and increases microtubules. Changes to Pericentrin and microtubules mislocalizes centriolar satellites in a pericentrosomal crowd.

cell biology↗

bioRxiv2021

Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

cell biology↗

bioRxiv2020

Single cell RNA analysis of trunk neural crest cells in zebrafish identifies pre-migratory populations expressing markers of differentiated derivatives

The neural crest is a migratory population of stem-like cells that contribute to multiple traits including the bones of the skull, peripheral nervous system, and pigment. How neural crest cells differentiate into diverse cell types is a fundamental question in the study of vertebrate biology. Here, we use single cell RNA sequencing to characterize transcriptional changes associated with neural crest cell development in the zebrafish trunk during the early stages of migration. We show that neural crest cells are transcriptionally diverse, and identify pre-migratory populations already expressing genes associated with differentiated derivatives. Further, we identify a population of Rohon-Beard neurons that are shown to be sources of Fgf signaling in the zebrafish trunk. The data presented identify novel genetic markers for multiple trunk neural crest cell populations and Rohon-Beard neurons providing insight into previously uncharacterized genes critical for vertebrate development.

developmental biology↗

bioRxiv2020

The Rab11 effectors Fip5 and Fip1 regulate zebrafish intestinal development

The Rab11 apical recycling endosome pathway is a well-established regulator of polarity and lumen formation; however, Rab11-vesicular trafficking also directs a diverse array of other cellular processes, raising the question of how Rab11 vesicles achieve specificity in space, time, and content of cargo delivery. In part, this specificity is achieved through effector proteins, yet the role of Rab11 effector proteins in vivo remains vague. Here, we use CRISPR/Cas9 gene editing to study the role of the Rab11 effector Fip5 during zebrafish intestinal development. Zebrafish contain two paralogous genes, fip5a and fip5b, that are orthologs of human FIP5. We find that fip5a and fip5b mutant fish show phenotypes characteristic of microvillus inclusion disease, including microvilli defects, inclusion bodies, and lysosomal accumulation. Single and double mutant analysis suggest that fip5a and fip5b function in parallel and regulate apical trafficking pathways required for assembly of keratin at the terminal web. Remarkably, in some genetic backgrounds, the absence of Fip5 triggers protein upregulation of a closely related family member, Fip1. This compensation mechanism occurs both during zebrafish intestinal development and in tissue culture models of lumenogenesis. In conclusion, our data implicate the Rab11 effectors Fip5 and Fip1 in a trafficking pathway required for apical microvilli formation.

cell biology↗

bioRxiv2020

Rab14/MACF2/CAMSAP3 Complex Regulates Endosomal Targeting to the Abscission Site During Cytokinesis

Abscission is complex cellular process that is required for mitotic division. It is well-established that coordinated and localized changes in actin and microtubule dynamics are vital for cytokinetic ring formation, as well as establishment of the abscission site. Actin cytoskeleton reorganization during abscission would not be possible without the interplay between Rab11- and Rab35-containing endosomes and their effector proteins, whose roles in regulating endocytic pathways at the cleavage furrow have now been studied extensively. Here, we identified Rab14 as novel regulator of abscission. We demonstrate that depletion of Rab14 causes either cytokinesis failure or significantly prolongs division time. We show that Rab14 regulates the efficiency of recruiting Rab11-endosomes to the central spindle microtubules and that Rab14 knockout leads to inhibition of actin clearance at the abscission site. Finally, we demonstrate that Rab14 binds to microtubule minus-end interacting MACF2/CAMSAP3 complex and that this binding is required for targeting of early endosomes to the central spindle. Collectively, our data identified Rab14/MACF2/CAMSAP3 as a protein complex that regulates Rab11-endosome targeting and the establishment of the abscission site.

cell biology↗

bioRxiv2019

CLIC4, a new component of the cytokinetic ring, regulates actin cytoskeleton dynamics during the anaphase-to-telophase transition

During mitotic cell division, the actomyosin cytoskeleton undergoes several dynamic changes that play key roles in progression through mitosis. While the regulators of cytokinetic ring formation and contraction are well-established, proteins that regulate cortical stability during anaphase and telophase have been understudied. Here, we describe a role for CLIC4 in regulating actin and actin-regulators at the cortex and cytokinetic cleavage furrow during cytokinesis. We first describe CLIC4 as a new component of the cytokinetic cleavage furrow that is required for successful completion of mitotic cell division. We also demonstrate that CLIC4 regulates the remodeling of sub-plasma membrane actomyosin network within the furrow by recruiting MST4 kinase and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in the transition from the contracting cytokinetic ring to the intercellular bridge during cytokinesis.

cell biology↗