Biology subjects

Bell, M.

Publications and source records attributed to Bell, M..

28 records · Page 2Linked to original sources

bioRxiv2020

Mechanical principles governing the shapes of dendritic spines

Dendritic spines are small, bulbous protrusions along the dendrites of neurons and are sites of excitatory postsynaptic activity. The morphology of spines has been implicated in their function in synaptic plasticity and their shapes have been well-characterized, but the potential mechanics underlying their shape development and maintenance have not yet been fully understood. In this work, we explore the mechanical principles that could underlie specific shapes using a minimal biophysical model of membrane-actin interactions. Using this model, we first identify the possible force regimes that give rise to the classic spine shapes - stubby, filopodia, thin, and mushroom-shaped spines. We also use this model to investigate how the spine neck might be stabilized using periodic rings of actin or associated proteins. Finally, we use this model to predict that the cooperation between force generation and ring structures can regulate the energy landscape of spine shapes across a wide range of tensions. Thus, our study provides insights into how mechanical aspects of actin-mediated force generation and tension can play critical roles in spine shape maintenance.

biophysics↗

bioRxiv2020

Axonal TAU sorting in SH-SY5Y-derived neurons requires the C-terminus of TAU but is independent of Ankyrin G

Somatodendritic missorting of the axonal protein TAU is a hallmark of Alzheimers disease and related tauopathies. Cultured rodent primary neurons and iPSC-derived neurons are used for studying mechanisms of neuronal polarity, including TAU trafficking. However, these models are expensive, time-consuming and/or require the sacrification of animals. In this study, we evaluated four differentiation procedures to generate mature neuron cultures from human SH-SY5Y neuroblastoma cells, in comparison to mouse primary neurons, and tested their TAU sorting capacity. We show that SH-SY5Y-derived neurons, differentiated with sequential RA/BDNF treatment, are suitable for investigating axonal TAU sorting. These human neurons show pronounced neuronal polarity, axodendritic outgrowth, expression of the neuronal maturation markers TAU and MAP2, and, importantly, efficient axonal sorting of endogenous and transfected human wild type TAU, similar to primary neurons. We demonstrate that axonal TAU enrichment requires the presence of the C-terminal half, as a C-terminus-lacking construct (N-term-TAUHA) is not axonally enriched in both neuronal cell models. Moreover, SH-SY5Y-derived neurons do not show formation of a classical axon initial segment (AIS), indicated by the lack of Ankyrin G (ANKG) and tripartite motif-containing protein 46 (TRIM46) at the proximal axon, which suggests that successful axonal TAU sorting is independent of classical AIS formation. Taken together, our results suggest i) that SH-SY5Y-derived neurons are a valuable human neuronal cell model for studying TAU sorting, which is readily accessible at low cost and without animal need, and that ii) the mechanisms of axonal TAU targeting require the TAU C-terminal half but are independent of ANKG or TRIM46 enrichment at the proximal axon.

neuroscience↗

bioRxiv2020

Subcellular localization of TAU isoforms and their influence on microtubule dynamics

In the adult human brain, six isoforms of the microtubule-associated protein TAU are expressed, which result from alternative splicing of exons 2, 3 and 10 of the MAPT gene. These isoforms differ in the number of N-terminal inserts (0N, 1N, 2N) and C-terminal repeat domains (3R or 4R) and are differentially expressed depending on the brain region and developmental stage. Although all TAU isoforms can aggregate and form neurofibrillary tangles, some tauopathies, such as Picks Disease and Progressive Supranuclear Palsy, are characterized by the accumulation of specific TAU isoforms. The influence of the individual TAU isoforms in a cellular context, however, is understudied. In this report, we investigated the subcellular localization of the human-specific TAU isoforms in primary neurons, and analyzed TAU isoform-specific effects on cell area and microtubule dynamics in SH-SY5Y neuroblastoma cells. Our results show that 2N-TAU isoforms are particularly retained from axonal sorting and that axonal enrichment is independent from the number of repeat domains, but that the additional repeat domain of 4R-TAU isoform results in a general reduction of cell size and an increase of microtubule counts in cells expressing 4R-TAU isoforms. Our study points out that individual TAU isoforms may influence microtubule dynamics differentially both by different sorting patterns as well as by direct effects on microtubule dynamics.

neuroscience↗

bioRxiv2020

Tumor-infiltrating nerves create an electro-physiologically active microenvironment and contribute to treatment resistance

Patients with densely innervated tumors do poorly as compared to those with sparsely innervated disease. Why some tumors heavily recruit nerves while others do not, remains unknown as does the functional contribution of tumor-infiltrating nerves to cancer. Moreover, while patients receive chemotherapeutic treatment, whether these drugs affect nerve recruitment has not been tested. Using a murine model of ovarian cancer, we show that tumor-infiltrating sensory nerves potentiate tumor growth, decrease survival, and contribute to treatment resistance. Furthermore, matched patient samples show significantly increased tumor innervation following chemotherapy. In vitro analysis of tumor-released extracellular vesicles (sEVs) shows they harbor neurite outgrowth activity. These data suggest that chemotherapy may alter sEV cargo, endowing it with robust nerve recruiting capacity.

cancer biology↗

bioRxiv2020

Genetically Encoded, Multivalent Liquid Glycan Array (LiGA)

The Central Dogma of Biology does not allow for the study of glycans using DNA sequencing. We report a "Liquid Glycan Array" (LiGA) platform comprising a library of DNA barcoded M13 virions that display 30-1500 copies of glycans per phage. A LiGA is synthesized by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins such as CD22 on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identifies the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo; measurements that cannot be performed with canonical glass slide-based glycan arrays. DedicationThe paper is dedicated to Laura L. Kiessling on the occasion of her 60th birthday.

biochemistry↗

bioRxiv2020

ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [89Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3-(A431) and GPC3+ cell lines including an engineered line (A431-GPC3+, G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [89Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [89Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [89Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

cancer biology↗

bioRxiv2019

Ecological and dietary differences between Ugandan chimpanzee communities with possible implications on tool use

Some East African chimpanzee (Pan troglodytes schweinfurthii) communities, such as the Sonso chimpanzees, display an unusually limited range of tool-use, but it remains unclear whether this is due to ecological and/or cultural factors. Information on ecological conditions and the diet of the Sonso chimpanzees in relation to neighbouring communities is needed. Here, we studied three adjacent communities in Budongo Forest (Sonso, Waibira, and Kamira), and the presumed core area of an undescribed community (Mwera), in the neighbouring Bugoma Forest. Through line-transects, we investigated (i) whether there were differences in food diversity and abundance between the communities home ranges; (ii) whether the home ranges differed in abundance of sticks and insect nests; and (iii) whether Sonso and Mwera chimpanzees differed in their diet (using faecal samples). Across communities, Sonso had the richest food availability and the lowest insect nest abundance. However, food availability in Mwera, Bugoma, was richer than Budongo communities that neighbour the Sonso territory, suggesting that there may be variation within Budongo. Data from faecal samples replicated our direct observations of food availability suggesting that Sonso chimpanzees had a broader diet than Mwera chimpanzees. This difference in foods availability may partially explain the Sonso chimpanzees lack of stick-tool-use, and low levels of insectivory. The tool repertoire of the other communities is currently unknown; however, we make predictions based on our ecological data. More detailed knowledge of small-scale variation in ecology within and between forest habitats may be important to advancing our understanding of the drivers of tool-use.\n\nSIGNIFICANCE STATEMENT - HIGHLIGHTED STUDENT PAPERTo advance our knowledge of the role of ecological factors in the emergence of tool use in chimpanzees, a nuanced understanding of the ecological conditions different chimpanzee communities experience is needed. We studied four Ugandan chimpanzee communities in two forests. One of these communities, Sonso, in the Budongo Forest, is well-known for its restricted range of tool types, including a total absence of stick use. Food diversity and abundance were highest, and stick tool use opportunities (abundance of sticks and insect nests) were lowest for the core-habitat of the Sonso chimpanzees in contrast to the other communities. We argue that ecological factors play a role in their unusual pattern of tool use, and make predictions about the expected types of tool use in the other communities based on their ecology. Thus, our study provides information that may help advance our understanding of how tool use arises under varied socioecological circumstances.

ecology↗

bioRxiv2019

DECODING THE MECHANOCHEMISTRY OF NEURITE PLASTICITY

The formation and disruption of synaptic connections during development is a fundamental step in neural circuit formation. Subneuronal structures such as neurites are known to be sensitive to the level of spontaneous neuronal activity but the specifics of how neurotransmitter-induced calcium activity regulates neurite homeostasis are not yet fully understood. In response to stimulation by neurotransmitters such as acetylcholine, calcium responses in cells are mediated the Gq/phospholipase C{beta} (PLC{beta})/ phosphatidylinositol 4,5 bisphosphate (PI(4, 5)P2) signaling pathway. Here, we show that prolonged Gq stimulation results in the retraction of neurites in PC12 cells and rupture of neuronal synapses by modulating membrane tension. To understand the underlying cause, we dissected the behavior of individual components of the Gq/PLC{beta}/PI(4, 5)P2 pathway during retraction, and correlated these to the retraction of the membrane and cytoskeletal elements impacted by calcium signaling. We developed a mathematical model that combines biochemical signaling with membrane tension and cytoskeletal mechanics, to show how signaling events are coupled to retraction velocity, membrane tension and actin dynamics. The coupling between calcium and neurite retraction is shown to be operative in the C. elegans nervous system. This study uncovers a novel mechanochemical connection between the Gq/PLC{beta}/(PI(4, 5)P2 pathway that couples calcium responses with neural plasticity.

biophysics↗

bioRxiv2019

A new class of disordered elements controls DNA replication through initiator self-assembly

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

biochemistry↗

bioRxiv2018

Dendritic spine geometry and spine apparatus organization govern the spatiotemporal dynamics of calcium

Dendritic spines are small subcompartments that protrude from the dendrites of neurons and are important for signaling activity and synaptic communication. These subcompartments have been characterized to have different shapes. While it is known that these shapes are associated with spine function, the specific nature of these shape-function relationships is not well understood. In this work, we systematically investigated the relationship between the shape and size of both the spine head and spine apparatus, a specialized endoplasmic reticulum compartment in the spine head, in modulating rapid calcium dynamics using mathematical modeling. We developed a spatial multi-compartment reaction-diffusion model of calcium dynamics in three dimensions with various flux sources including N-methyl-D-aspartate receptors (NMDAR), voltage sensitive calcium channels (VSCC), and different ion pumps on the plasma membrane. Using this model, we make several important predictions - first, the volume-to-surface area ratio of the spine regulates calcium dynamics, second, membrane fluxes impact calcium dynamics temporally and spatially in a nonlinear fashion, and finally the spine apparatus can act as a physical buffer for calcium by acting as a sink and rescaling the calcium concentration. These predictions set the stage for future experimental investigations of calcium dynamics in dendritic spines.

systems biology↗