Biology subjects

Allen, P.

Publications and source records attributed to Allen, P..

15 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↗

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↗

bioRxiv2024

Multiplexed Glycan Immunofluorescence Identification of Pancreatic Cancer Cell Subpopulations in Both Tumor and Blood Samples

Pancreatic ductal adenocarcinoma (PDAC) tumor heterogeneity impedes the development of biomarker assays suitable for early disease detection that would improve patient outcomes. The CA19-9 glycan is currently used as a standalone biomarker for PDAC. Furthermore, previous studies have shown that cancer cells may display aberrant membrane-associated glycans. We therefore hypothesized that PDAC cancer cell subpopulations could be distinguished by aberrant glycan signatures. We used multiplexed glycan immunofluorescence combined with pathologist annotation and automated image processing to distinguish between PDAC cancer cell subpopulations within tumor tissue. Using a training-set/test-set approach, we found that PDAC cancer cells may be identified by signatures comprising 4 aberrant glycans (VVL, CA19-9, sTRA, and GM2) and that there are three glycan-defined PDAC tumor types: sTRA type, CA19-9 type, and intermixed. To determine whether the aberrant glycan signatures could be detected in blood samples, we developed hybrid glycan sandwich assays for membrane-associated glycans. In both patient-matched tumor and blood samples, the proportion of aberrant glycans detected was consistent. Furthermore, our multiplexed glycan immunofluorescent approach proved to be more sensitive and more specific than CA19-9 alone. Our results provide proof of concept for a novel methodology to improve early PDAC detection and patient outcomes.

cancer biology↗

bioRxiv2024

Transcriptional and neurochemical signatures of cerebral blood flow alterations in schizophrenia and the clinical high-risk state for psychosis

The brain integrates multiple scales of description, from the level of cells and molecules to large-scale networks and behaviour, and understanding the relationships between these layers may be fundamental to advancing our understanding of how the brain works in health and disease. Recent neuroimaging research has shown that alterations in brain function that are associated with schizophrenia spectrum disorders (SSD) are already present in young adults at clinical high-risk for psychosis (CHR-P), yet the cellular and molecular determinants of these alterations are not well understood. Here, combining regional cerebral blood flow (rCBF) data with existing transcriptomic and neurotransmitter data, we show that cell-types involved in stress response and inflammation, as well as the dopamine, acetylcholine, GABAA and NMDA receptor systems, align as shared and distinct cellular and neurochemical signatures of rCBF phenotypes in people with SSD and those at CHR-P. Decoding the biological pathways involved in neuroimaging-based psychosis phenotypes may provide a basis for the development of novel interventions.

neuroscience↗

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↗

bioRxiv2023

Oral administration of GnRH via a cricket vehicle stimulates spermiation in tiger salamanders (Ambystoma tigrinum)

More than 50% of caudates are threatened with extinction and are in need of ex-situ breeding programs to support conservation efforts and species recovery. Unfortunately, many salamander populations under human care can experience reproductive failure, primarily due to missing environmental cues necessary for breeding. Assisted reproductive technologies (ARTs) are a useful suite of techniques for overcoming or bypassing these missing environmental cues to promote breeding. Exogenous hormones are used to stimulate natural breeding behaviors or gamete expression for in-vitro fertilization or biobanking and are typically administered intramuscularly in caudates. While effective, intramuscular injection is risky to perform in smaller-bodied animals, resulting in health and welfare risks. This research investigated the spermiation response to hormone administration through a non-invasive oral route using the tiger salamander (Ambystoma tigrinum) as a model species. Male salamanders were randomly rotated six weeks apart through four treatments (n = 11 males/treatment) in which animals received a resolving dose of gonadotropin-releasing hormone (GnRH) as follows: (1) Prime-Only (0.0 {micro}g/g); (2) Low (0.25 {micro}g/g); (3) Medium (1.0 {micro}g/g); and (4) High (2.0 {micro}g/g). All males were given a GnRH priming dose (0.25 {micro}g/g) 24 hours prior to the resolving dose. Exogenous hormone was delivered inside of a cricket (Gryllodes sigillatus) that was presented as a food item by tweezers. Sperm samples were collected at 1, 3, 6, 9, 12, and 24 hours after the resolving dose and analyzed for quantity and quality. For all treatments, sperm concentration was produced in an episodic pattern over time. The Prime-Only treatment had a lower (p < 0.05) percent of sperm exhibiting normal morphology compared to treatments utilizing a resolving dose of GnRH. Overall, oral administration of GnRH is a feasible route of inducing spermiation in salamanders, yielding sperm of sufficient quantity and quality for in-vitro fertilization and biobanking efforts.

zoology↗

bioRxiv2023

RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1000nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with different RNA structures support a model where multiple weak interactions, such as with the CTD or the NTDs secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N. SignificanceThe nucleocapsid protein of the SARS-CoV-2 coronavirus binds to viral RNA, both to protect and condense it inside the viral particle and to facilitate viral transcription inside infected host cells. Evidence suggests that variations in RNA structure impact how and where it binds to the nucleocapsid, but these differences are not well understood at a structural level. Using nuclear magnetic resonance spectroscopy, we examine the interactions between each folded domain of the nucleocapsid and different RNA structures. Binding affinities and NMR chemical shift profiles demonstrate that binding between the N-terminal domain and single stranded RNA is driven by strong interactions at a specific site, while multiple weak nonspecific interactions at newly discovered sites lead to phase separation and RNA condensation.

biophysics↗

bioRxiv2023

Why flying insects gather at artificial light

For millennia, humans have watched nocturnal insects flying erratically around fires and lamps. Explanations have included theories of "lunar navigation" and "escape to light". However, without three-dimensional flight data to test them rigorously, this odd behaviour has remained unexplained. We employed high-resolution motion capture in the laboratory and stereo-videography in the field to reconstruct the 3D kinematics of insect flights around artificial lights. Contrary to the expectation of attraction, insects do not steer directly toward the light. Instead, insects turn their dorsum toward the light, generating flight bouts perpendicular to the source. Under natural sky light, tilting the dorsum towards the brightest visual hemisphere helps maintain proper flight attitude and control. Near artificial sources, however, this highly conserved dorsal-light-response can produce continuous steering around the light and trap an insect. Our guidance model demonstrates that this dorsal tilting is sufficient to create the seemingly erratic flight paths of insects near lights and is the most plausible model for why flying insects gather at artificial lights.

animal behavior and cognition↗

bioRxiv2023

Heterogeneity of Glycan Biomarker Clusters as an Indicator of Recurrence in Pancreatic Cancer

Outcomes following tumor resection vary dramatically among patients with pancreatic cancer. A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block tumor sections. The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.

cancer biology↗

bioRxiv2020

Daily and Intermittent Smoking Decrease Gray Matter Volume and Concentrations of Glutamate, Creatine, Myo-Inositol and N-acetylaspartate in the Prefrontal Cortex

Cigarette smoking is still the largest contributor to disease and death worldwide. Successful cessation is hindered by decreases in prefrontal glutamate concentrations and gray matter volume due to daily smoking. Because non-daily, intermittent smoking also contributes greatly to disease and death, understanding whether infrequent tobacco use is associated with reductions in prefrontal glutamate concentrations and gray matter volume may aid public health. Eighty-five young participants (41 non-smokers, 24 intermittent smokers, 20 daily smokers, mean age ~23 years old), underwent 1H-magnetic resonance spectroscopy of the medial prefrontal cortex, as well as structural MRI to determine whole-brain gray matter volume. Compared to non-smokers, both daily and intermittent smokers exhibited lower concentrations of glutamate, creatine, N-acetylaspartate and myo-inositol in the medial prefrontal cortex, and lower gray matter volume in the right inferior frontal gyrus; these measures of prefrontal metabolites and structure did not differ between daily and intermittent smokers. Finally, medial prefrontal metabolite concentrations and right inferior frontal gray matter volume were positively correlated, but these relationships were not influenced by smoking status. This study provides the first evidence that both daily and intermittent smoking are associated with low concentrations of glutamate, creatine, N-acetylaspartate and myo-inositol, and low gray matter volume in the prefrontal cortex. Future tobacco cessation efforts should not ignore potential deleterious effects of intermittent smoking by considering only daily smokers. Finally, because low glutamate concentrations hinder cessation, treatments that can normalize tonic levels of prefrontal glutamate, such as N-acetylcysteine, may help intermittent and daily smokers to quit.

neuroscience↗

bioRxiv2020

Detection of Chemotherapy-Resistant Pancreatic Cancer Using a Glycan Biomarker

Background and AimsA subset of pancreatic ductal adenocarcinomas (PDACs) is highly resistant to systemic chemotherapy, but no markers are available in clinical settings to identify this subset. We hypothesized that chemotherapy-resistant PDACs express a glycan biomarker called sTRA. Methods. We tested this marker to identify treatment-resistant PDAC in multiple systems: sets of cell lines, organoids, and isogenic cell lines; primary tumors; and blood plasma from cohorts of human subjects. Results. Among a panel of 27 cell lines, high levels of cell-surface sTRA identified higher resistance to seven chemotherapeutics used against PDAC. Using primary tumors from two different cohorts, patients who were positive for a gene-expression classifier for sTRA received no statistically significant benefit from adjuvant chemotherapy, in contrast to those negative for the signature. In another cohort, using direct measurements of sTRA in tissue microarrays by quantitative immunofluorescence, patients who were high in sTRA again had no statistically significant benefit from adjuvant chemotherapy. Further, a blood-plasma test for the sTRA glycan identified the PDACs that showed rapid relapse following neoadjuvant chemotherapy. This blood test performed with 96% specificity and 56% sensitivity in a blinded cohort using samples collected before the start of treatment. Conclusion. These findings establish that tissue or plasma sTRA can identify PDACs that are resistant to neoadjuvant or adjuvant chemotherapy. This capability could help apply systemic treatments more precisely and facilitate biomarker-guided trials targeting resistant PDAC.

cancer biology↗

bioRxiv2020

Did I imagine that? The functional role of paracingulate cortex in reality monitoring

Reality monitoring describes our ability to distinguish between internally and externally generated experiences. Individuals show significant variation in this ability and impaired reality monitoring has been linked to the experience of hallucinations. We undertook two studies to investigate the association between reality monitoring and morphology of the paracingulate region of medial prefrontal cortex. In Study 1 we compared reality monitoring accuracy and functional connectivity within paracingulate cortex in groups of healthy controls (N=20) and patients with schizophrenia and hallucinations (N=19). Controls showed greater reality monitoring accuracy that was associated with resting-state functional connectivity between paracingulate, precuneus and occipital cortices, while reality monitoring in patients was associated with more lateral functional connectivity. In Study 2 we used real-time fMRI neurofeedback to obtain causal evidence for the role of the paracingulate cortex in reality monitoring. Healthy individuals received Active feedback from paracingulate cortex (N=21) or Sham feedback based on randomised signal (N=18). Active-group participants showed a specific behavioural effect of improved reality monitoring for Imagined items, as well as increases in both activity within the paracingulate region, and its posterior functional connectivity with precuneus and lateral parietal cortices, and occipital cortex. Our findings suggest reality monitoring in healthy individuals is causally supported by a paracingulate mediated flexible network including the precuneus. Network connectivity can be enhanced using neurofeedback and tracks with improved reality monitoring ability. In contrast, patients with schizophrenia may utilise a distinct and more lateral network which may explain observed sub-optimal reality monitoring accuracy, contributing to the experience of hallucinations. Significance StatementReality monitoring refers to our ability to distinguish imagination from our experiences in the outside world, and is linked both to hallucinations in schizophrenia as well as to the morphology of paracingulate cortex area of the brain. Here, we revealed less paracingulate involvement in the functional reality monitoring networks in patients with schizophrenia compared to healthy individuals. Thereafter, we used real-time fMRI neurofeedback to show that healthy individuals can learn to upregulate brain activity within the paracingulate cortex, with this resulting in both improved reality monitoring ability and changes in paracingulate functional connectivity. This suggests that paracingulate cortex activity and connectivity play a causal role in reality monitoring, with implications for both the understanding and treatment of hallucinations.

neuroscience↗

bioRxiv2018

Dynamics of spontaneous alpha activity correlate with language ability in young children

Early childhood is a period of tremendous growth in both language ability and brain maturation. To understand the dynamic interplay between neural activity and spoken language development, we used resting-state EEG recordings to explore the relation between alpha oscillations (7-10 Hz) and oral language ability in 4- to 6-year-old children with typical development (N=41). Three properties of alpha oscillations were investigated: a) alpha power using spectral analysis, b) flexibility of the alpha frequency quantified via the oscillation's moment-to-moment fluctuations, and c) scaling behavior of the alpha oscillator investigated via the long-range temporal correlation in the alpha-amplitude time course. All three properties of the alpha oscillator correlated with children's oral language abilities. Higher language scores were correlated with lower alpha power, greater flexibility of the alpha frequency, and longer temporal correlations in the alpha-amplitude time course. Our findings demonstrate a cognitive role of several properties of the alpha oscillator that has largely been overlooked in the literature.

neuroscience↗

bioRxiv2017

A persistent lack of International representation on editorial boards in biology

The scholars comprising journal editorial boards play a critical role in defining the trajectory of knowledge in their field. Nevertheless, studies of editorial board composition remain rare, especially those focusing on journals publishing research in the increasingly globalized fields of science, technology, engineering, and math (STEM). Using metrics for quantifying the diversity of ecological communities, we quantified international representation on the 1985-2014 editorial boards of twenty-four environmental biology journals. Over the course of three decades there were 3831 unique scientists based in 70 countries that served as editors. The size of the editorial community increased over time - there were 420% more editors serving in 2014 than in 1985 - as did the number of countries in which editors were based. Nevertheless, editors based outside the Global North (the group of economically developed countries with high per capita Gross Domestic Product (GDP) that collectively concentrate most global wealth) were extremely rare. Furthermore, 67.06% of all editors were based in either the USA or UK. Consequently, Geographic Diversity - already low in 1985 - remained unchanged through 2014. We argue that this limited geographic diversity can detrimentally affect the creativity of scholarship published in journals, the progress and direction of research, the composition of the STEM workforce, and the development of science in Latin America, Africa, the Middle East, and much of Asia (i.e., the Global South).

scientific communication and education↗