Biology subjects

Weems, A. D.

Publications and source records attributed to Weems, A. D..

7 recordsLinked to original sources

bioRxiv2026

Septin inhibitors exert opposing effects on distinct higher-order septin structures in melanoma cells

Septins are cytoskeletal GTPases that assemble into diverse higher-order structures with context-dependent mechanical and signaling functions. Recent work identified a septin-dependent pro-survival pathway in metastatic cancer cells, termed bleb signaling, in which plasma membrane blebbing promotes the formation of septin signaling hubs that support survival during cellular detachment and dissemination. Disrupting these hubs may offer a novel therapeutic strategy targeting disseminated cancer cells. Here, we compare the effects of three septin-targeting compounds, forchlorfenuron (FCF), UR214-9, and REM127, on septin organization and viability in melanoma cells using confocal microscopy and quantitative computer vision analysis. FCF and UR214-9 both effectively destabilized septin hubs, while REM127 had no appreciable effect. FCF also reproduced the context-specific viability reduction previously seen in detached melanoma cells, while UR214-9 caused broad off-target toxicity and cell death in all growth conditions. As with hub disruption, REM127 produced no effect on viability. Surprisingly, though none of the compounds caused an increase in higher-order septin structures during detachment, all three increased end-on-end polymerization of filamentous septins at stress fibers when grown adhered to substrate. These findings demonstrate that septin-targeting compounds exert divergent effects on different classes of septin higher-order structures within the same cell type, promoting filamentous assembly while disrupting or failing to promote membrane-associated signaling hubs. This highlights not only the structural distinctiveness of such classes, but that pharmacological targeting can produce opposite effects on them. Moreover, this study emphasizes the need for next-generation septin inhibitors capable of selectively targeting disease-relevant septin assemblies.

cell biology↗

bioRxiv2026

In vivo evidence for bleb-induced survival signaling in metastatic melanoma

Bleb signaling is a cellular process in which pressure-driven plasma membrane protrusions generate localized micron-scale membrane curvature that recruits cytosolic septin complexes, assembling signaling hubs that promote cell survival1. Our prior work showed that this morphology-encoded signaling pathway is necessary to sustain anchorage-independent survival of BRAF and NRAS mutant melanoma cells in vitro. However, whether bleb-induced signaling occurs in vivo and contributes to cancer progression is unclear. Here, we develop complementary in vivo and ex vivo assays spanning human patient samples and mouse xenograft models to mechanistically interrogate bleb signaling directly in physiologic contexts. We observe that bleb-associated septin hubs are exclusively formed in poorly adherent amoeboid tumor cells at the invasive margin, within malignant effusions, and at distant metastatic sites, while well-adhered cells in the tumor interior show no signs of septin hub formation. Accordingly, pharmacological pathway disruption specifically kills disseminated tumor cells within these low-adhesion microenvironments while having no appreciable effect on adhered cells in the tumor interior, resulting in reduced metastatic burden and delayed disease recurrence in vivo. This work confirms septin-mediated bleb signaling as a previously unrecognized vulnerability of disseminated cancer cells and demonstrates that this morphology-encoded pathway operates in vivo to support disease progression, preserving cancer cell viability under conditions in which cell survival signals from the environment are muted. These findings suggest novel opportunities to target survival signaling in micrometastatic disseminated cancer cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/697601v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1770384org.highwire.dtl.DTLVardef@87e567org.highwire.dtl.DTLVardef@1e5d6b2org.highwire.dtl.DTLVardef@724c5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Blebs Promote Cell Survival by Assembling Oncogenic Signaling Hubs

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signaling pathways, without which cells undergo anoikis - a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cancer cells often lose firm attachment to surrounding tissue2-5. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs6-13. Bleb function has been thoroughly investigated in the context of amoeboid migration but is far less examined in other scenarios14-19. Here we show by quantitative subcellular 3D imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signaling hubs that confer anoikis resistance. Specifically, we discovered in melanoma cells that blebbing generates plasma membrane contours, which recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signaling hubs activate ERK and PI3K - canonical promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils an unanticipated morphological requirement for mutant NRAS to operate as an effective oncoprotein. Moreover, we find that though some BRAF mutant melanoma do not rely on this survival pathway in a basal state, BRAF/MEK inhibition strongly sensitizes them to both bleb and septin inhibition. Importantly, we demonstrate that fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harboring oncogenic mutations. These data define a role for blebs as potent signaling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

cell biology↗

bioRxiv2021

Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach to identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Chaperone inhibition liberates the mutant to enter the nucleus where it has a slight dominant-negative effect. These findings provide new insights into the effects of missense mutations.

genetics↗

bioRxiv2020

Worrying drives cell migration in mechanically unrestrained environments

Migratory cells often encounter crowded microenvironments through which they must find or make a path. Amoeboid cells are thought to find a path by deforming their bodies to squeeze through tight spaces. Yet many amoeboid cells seem to maintain a near spherical morphology as they move. To examine this unexplored mechanism of migration, we visualized amoeboid melanoma cells in dense environments and found that they carve a path via bleb-driven mechanical degradation of extracellular matrix components without proteolytic degradation. Interactions between adhesions and collagen at the cell front induce a signaling cascade that promotes bleb enlargement via branched actin polymerization. Large blebs abrade collagen, creating feedback between extracellular matrix structure, cell morphology and polarization that enables both path generation and persistent movement.

cell biology↗

bioRxiv2019

Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast

Septin proteins co-assemble into hetero-oligomers that polymerize into cytoskeletal filaments with a variety of cellular functions. In Saccharomyces cerevisiae, where septins were first discovered, five subunits comprise two species of septin hetero-octamers, Cdc11/Shs1-Cdc12-Cdc3-Cdc10- Cdc10-Cdc3-Cdc12-Cdc11/Shs1. Septins evolved from ancestral GTPases. We previously found evidence that slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1 into septin complexes. It was unclear why many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that the small molecule guanidine hydrochloride (GdnHCl) rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1-Cdc12-Cdc3- Cdc3-Cdc12-Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium ion (Gdm) occupies the site of a "missing" Arg sidechain that is present in other fungal species in which (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers co-exist with octamers in wild-type cells. These findings support a model in which Gdm reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to hetero-octamers. Given that septin hexamers made natively in human cells also exclude Cdc10-like central subunits via homodimerization of an active GTPase, our results provide new mechanistic details that likely apply to septin assembly throughout phylogeny.

genetics↗

bioRxiv2019

A unified role for membrane-cortex detachment during cell protrusion initiation

Cell morphogenesis employs a diversity of membrane protrusions. They are discriminated by differences in force generation. Actin polymerization is the best studied mechanism of force generation, but growing interest in how variable molecular conditions and microenvironments alter morphogenesis has revealed other mechanisms, including intracellular pressure. Here, we show that local depletion of membrane cortex links is an essential step in the initiation of both pressure-based and actin-based protrusions. This observation challenges the quarter-century old Brownian ratchet model of actin-driven membrane protrusion, which requires an optimal balance of actin filament growth and membrane tethering. An updated model confirms membrane-filament detachment is necessary to activate the ratchet mechanism. These findings unify the regulation of different protrusion types, explaining how cells generate robust yet flexible strategies of morphogenesis.

cell biology↗