Biology subjects

Bergstralh, D. T.

Publications and source records attributed to Bergstralh, D. T..

10 recordsLinked to original sources

bioRxiv2026

Reconstruction of septin higher-order nano-size structures in ovarian cancer cells uncover susceptibility to the septin-targeting small molecule UR214-9

In cancer cells, septins assemble into enigmatic higher-order structures of 300-700 nanometers, including long needle-like filaments, thick perinuclear rings, and cytoplasmic bundles or aggregates. The absence of genetic or pharmacological tools to recapitulate these architectures in-vitro has impeded mechanistic studies of their formation, function, and therapeutic targeting. Here, first, determining the overexpression of septin-2 in epithelial ovarian cancer (EOC) and its association with increased mortalities and dependencies, we select SKOV-3 ovarian cancer cells as a tractable model in which septin supramolecular assemblies can be recreated in-vitro and interrogated. This system shows that the forchlorfenuron (FCF) analog UR214-9 remodels septin architecture, converting co-expressed human septin octamers (SEPT2-SEPT6-SEPT7-SEPT9-SEPT9-SEPT7-SEPT6-SEPT2) into large cytoplasmic aggregates. In parallel, transiently expressed SEPT2 is reorganized into septin-rich noodle-like filaments, perinuclear rings, and web-like networks encircling the nucleus upon UR214-9 treatment. Mechanistically, UR214-9 disrupts the incorporation of SEPT2, SEPT7, and SEPT9 into canonical septin hetero-octamers, resulting in assembly-defective or imperfect oligomers that preferentially reorganize into these aberrant higher-order structures. This aggregation likely prevents septin-2 migration during interphase-to-cleavage furrow transition in NRK-49F-SEPT2-EGFP homozygous cells and impacts SKOV-3 cytokinesis, cell proliferation, adhesion and invasion and migration while sparing ceramide transport to the Golgi, preserving ER and cis-Golgi structure. These effects manifested in reduced growth of ovarian, endometrial and breast cancer xenografts without attracting significant off-target engagements per the global transcriptomic analysis of JIMT1 breast cancer and PANC-1 pancreatic cells. UR214-9 treated animals showed observable safety in animals. Thus, a tool to recreate aberrant septin structures and identification of septins as a druggable cytoskeletal target for ovarian, endometrial, breast and pancreatic cancer by perturbing their hetero-octamerization assembly is presented. SignificanceWe provide a method to reconstruct the higher-order septin architecture observed in cancer cells, to study their assembly and functions. Intriguingly, cancer cells tolerate hetero-oligomeric septins lacking specific subunits, suggesting that compositionally deficient oligomers are not efficiently targeted for degradation, unlike unincorporated septin monomers in normal cells. This tolerance may enable accumulation of structurally aberrant septin complexes acquiring long-needles, rings or thick-aggregates in disease cells. We further show that septin oligomerization can be pharmacologically perturbed. By integrating structural, cellular, and energetic readouts using in-silico techniques, we establish a quantitative framework for septin-targeted modulation, generating UR214-9 as a new chemotype that disrupts septin oligomeric assembly via preventing incorporation of SEPT2/7/9, into canonical hetero-octamers, causes defects in cytokinesis, altered cell migration, viability, and remodels septin-actin architectures, ultimately impairing tumor cell growth. Thus, pharmacological targeting of septin assembly represents a tractable strategy to perturb septin-dependent cellular processes in cancer and neurodegenerative diseases with reported septin dysregulation.

cancer biology↗

bioRxiv2026

Fasciclin 2 Cooperates with Discs Large to Maintain Epithelial Architecture

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) coordinate adhesive interactions with intracellular organization during tissue morphogenesis. In the Drosophila follicular epithelium, epithelial maintenance depends on reintegration, a process in which mitotically displaced cells reincorporate into the epithelial monolayer. Previous work identified the IgCAMs Fasciclin 2 (Fas2) and Neuroglian (Nrg) as parallel, partially redundant regulators of reintegration, but the intracellular mechanisms linking adhesion to reintegration remained unclear. Here, we show that Fas2 supports reintegration through two mechanistically distinct modes: a transmembrane mode and a GPI-linked mode. Although both contribute to reintegration, the transmembrane mechanism is more effective and depends on stabilization of a cortical Fas2 pool through intracellular coupling. Using yeast two-hybrid screening, genetics, and fluorescence recovery after photobleaching (FRAP), we identify the scaffold protein Discs large (Dlg1) as a functional intracellular partner of transmembrane Fas2. Partial disruption of Dlg1 preferentially sensitizes epithelia in which the parallel Nrg-dependent reintegration mechanism is compromised, consistent with Dlg1 functioning primarily within the Fas2-dependent reintegration arm. While Dlg1 is not required for Fas2 membrane localization, Dlg1 disruption increases the mobile fraction of transmembrane Fas2, indicating that Dlg1 promotes retention of a stabilized cortical Fas2 pool. Together, these findings support a model in which epithelial reintegration depends on coordinated adhesion-scaffold coupling and reveal mechanistic parallels between epithelial reintegration and IgCAM-dependent processes in the developing nervous system.

cell biology↗

bioRxiv2025

Beyond pulling: microtubule pushing forces contribute to robust spindle orientation in regular and irregular cell shapes

Oriented cell division is fundamental to development and tissue organization, requiring precise control of both spindle positioning and orientation. While cortical pulling forces mediated by dynein motor proteins are well-established drivers of spindle dynamics, the contribution of microtubule polymerization-based pushing forces remains unclear. We developed a generalizable computational biophysical model that integrates both pulling and pushing mechanisms to investigate spindle behavior across diverse cell types and geometries. This model successfully recapitulates experimental observations in three well-studied models: Drosophila follicular epithelial cells, Drosophila neuroblasts, and the early C. elegans embryo. Systematic analysis reveals that while pulling forces are the primary drivers of directed spindle orientation, pushing forces play crucial supporting roles by preventing spindle stalling and promoting alignment dynamics, particularly at high initial misalignment angles. We further applied our model to irregularly shaped zebrafish endothelial cells, which present unique challenges due to their non-spherical morphology and dynamic shape changes during mitosis. Our results demonstrate that asymmetric cortical force generator distributions, potentially localized at cell-cell junctions, can account for the observed off-center spindle positioning in these cells. This work provides a unified framework for understanding how the interplay between cell geometry, molecular polarity cues, and competing physical forces determines spindle dynamics, offering new insights into both canonical and non-canonical division orientations across cell types.

biophysics↗

bioRxiv2024

Fas2: A Tale of Two Chromosomes

The cell-cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily-conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2EB112 has been used in at least 37 research articles, and Fas2G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2G0336) or enhances (Fas2EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg14, also a classic null allele.

genetics↗

bioRxiv2023

The Mechanical Influence of Densification on Initial Epithelial Architecture

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayer epithelium - whether protective, secretory, absorptive, or filtrative -relies on regular tissue architecture with respect to the apical-basal axis. Using an unbiased 3D analysis pipeline developed in our lab, we previously showed that epithelial tissue architectures in culture can be divided into distinct developmental categories, and that these are intimately connected to cell density: at sparse densities, cultured epithelial cell layers have a squamous morphology (Immature); at intermediate densities, these layers develop lateral cell-cell borders and rounded cell apices (Intermediate); cells at the highest densities reach their full height and demonstrate flattened apices (Mature). These observations prompted us to ask whether epithelial architecture emerges from the mechanical constraints of densification, and to what extent a hallmark feature of epithelial cells, namely cell-cell adhesion, contributes. In other words, to what extent is the shape of cells in an epithelial layer a simple matter of sticky, deformable objects squeezing together? We addressed this problem using a combination of computational modeling and experimental manipulations. Our results show that the first morphological transition, from Immature to Intermediate, can be explained simply by cell crowding. Additionally, we identify a new division (and thus transition) within the Intermediate category, and find that this second morphology relies on cell-cell adhesion.

cell biology↗

bioRxiv2022

The spindle orienting machinery requires activation, not just localization

The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Partner of Inscuteable (Pins), Gi, Mushroom body defective (Mud), and Dynein in flies - that exert a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In Drosophila neuroblasts, for example, this cue is thought to be Inscuteable, which helps recruit Pins to the apical cell surface. In this study we revisit the canonical model. We find that spindle orientation in the follicular epithelium requires not only Pins localization but also activation, which relies on direct interaction between Pins and the multifunctional protein Discs large. This mechanism is distinct from the one mediated by Inscuteable, which we find also has an activating step. Together our results show that the canonical model is incomplete. Local enrichment of Pins is not sufficient to determine spindle orientation; an activation step is also necessary.

cell biology↗

bioRxiv2021

Competition Between Cell-Cell and Cell-Substrate Adhesion Determines Epithelial Monolayer Architecture in Culture

Organ surfaces are lined by epithelial monolayers - sheets of cells that are one-cell thick. This architecture underlies tissue function, and its loss is associated with disease, including cancer. Studies of in-plane epithelial cell behaviors show that a developing epithelium behaves as a fluid in respect to the tissue plane, and can therefore readily adapt to varying mechanical influences during morphogenesis. We asked the question of how monolayer architecture is achieved, and whether it demonstrates the same fluid behavior. To address this problem, we cultured MDCK (Madin-Darby Canine Kidney) cell layers at different densities and timepoints and analyzed their architectures using a novel tool, Automated Layer Analysis (ALAn), which we introduce here. Our experimental and theoretical results lead us to propose that epithelial monolayer architecture is governed by a balance of counteracting forces due to cell-cell and cell-substrate adhesion, and that this balance is influenced by cell density. MDCK cells do not undergo obvious rearrangement along the apical-basal axis; instead, cells that do not contact the substrate aggregate on top of the monolayer. Our findings therefore imply that monolayered architecture is under more rigid control than planar tissue shape in epithelia.

cell biology↗

bioRxiv2021

Interaction between Discs large and GPSM2: A Comparison Across Species

The orientation of the mitotic spindle determines the direction of cell division, and therefore contributes to tissue shape and cell fate. Interaction between the multifunctional scaffolding protein Discs large (Dlg) and the canonical spindle orienting factor GPSM2 (also called Pins in Drosophila and LGN in vertebrates) has been established in bilaterian models, but its function remains unclear. We used a phylogenetic approach to test whether the interaction is obligate in animals, and in particular whether GPSM2 evolved in multicellular organisms as a Dlg-binding protein. We show that Dlg diverged in C. elegans and the syncytial sponge O. minuta and propose that this divergence may correspond to differences in spindle orientation requirements between these organisms and the canonical pathways described in bilaterians. We also demonstrate that GPSM2 is present in basal animals, but the established Dlg-interaction site cannot be found in either Placozoa or Porifera. Our results suggest that the interaction between GPSM2 and Dlg appeared in Cnidaria, and we therefore speculate that it may have evolved to promote accurate division orientation in the nervous system. This work reveals the evolutionary history of the GPSM2/Dlg interaction and suggests new possibilities for its importance in spindle orientation during epithelial and neural tissue development.

cell biology↗

bioRxiv2020

An Axon-Pathfinding Mechanism Preserves Epithelial Tissue Integrity

Dividing cells often move apically within epithelial tissue layers, likely to escape the spatial confinement of their neighbors. Because of this movement, daughter cells may be born displaced from the tissue layer. Reintegration of these displaced cells helps support tissue growth and maintain tissue architecture. In the Drosophila follicular epithelium, reintegration relies on the immunoglobulin-superfamily cell-adhesion molecules (IgCAMs) Neuroglian and Fasciclin 2, which line cell-cell borders1. These molecules have been described in epithelia, but are well-studied for their roles in neural development2-8. We show here that reintegration works in the same way as IgCAM-mediated axon growth and pathfinding; it relies not only on extracellular adhesion but also mechanical coupling between IgCAMs and the lateral Spectrin-Based Membrane Skeleton. Our work indicates that reintegration is mediated by a distinct epithelial cell-cell junction that is compositionally and functionally equivalent to junctions made between axons.

cell biology↗

bioRxiv2017

Bimodal Spindle Orientation Drives Tissue Regularity in a Proliferating Epithelium

We investigated the relationship between proliferation and tissue topology in an epithelial tissue undergoing elongation. We found that cell division is not required for elongation of the early Drosophila follicular epithelium, but does drive the tissue towards optimal geometric packing. To increase tissue regularity, cell divisions are oriented in the planar axis, along the direction of tissue expansion. Planar division orientation is governed by apico-cortical tension, which aligns with tissue expansion but not with interphase cell shape elongation. Hertwigs Rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. We tested whether this observation could be explained by anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis. We found that Pins/Mud does not participate in planar division orientation. Rather, tension translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin to adherens junctions. These findings demonstrate that division orientation in different axes - apical-basal and planar - is controlled by distinct, independent mechanisms in a proliferating epithelium.\n\nSummary StatementRegularity in a proliferating epithelium requires cells to divorce division orientation from interphase shape, which they accomplish by using distinct mechanisms to orient divisions in the apical-basal and planar axes.

developmental biology↗