Biology subjects

Ivaska, J.

Publications and source records attributed to Ivaska, J..

8 recordsLinked to original sources

bioRxiv2026

Pancreatic cancer cells breach endothelial barriers through protrusion-driven invasion or endothelial retraction

Extravasation, the exit of circulating cancer cells from blood vessels, is a critical yet poorly understood step in metastatic dissemination. Here we show that pancreatic ductal adenocarcinoma (PDAC) cells can breach endothelial barriers through two mechanistically distinct modes of extravasation. MIA PaCa-2 cells breach endothelial junctions via filopodia-like protrusions, enabling access to and spread across the basal extracellular matrix (ECM). By contrast, AsPC-1 cells remain rounded atop the endothelium and cross the barrier by triggering rapid retraction of neighbouring endothelial cells. These distinct extravasation modes were also observed in zebrafish larvae. In the mouse lung, AsPC-1 cells arrest, survive, induce endothelial detachment from the basal lamina, and extravasate through this retraction mechanism before metastatic outgrowth. Mechanistically, AsPC-1-secreted factors are sufficient to destabilise endothelial monolayers, and AsPC-1 cells also induce endothelial apoptosis; however, blocking apoptosis does not prevent barrier disruption. By contrast, treatment with saracatinib, a Src-family kinase inhibitor, protects endothelial barriers, limits early vascular disruption in the lung, and delays metastatic outgrowth. Together, these findings reveal that PDAC cells can extravasate via mechanistically distinct routes, suggesting that effective anti-metastatic strategies may need to target multiple modes of endothelial barrier breach rather than a single pathway.

cell biology↗

bioRxiv2018

Superresolution architecture of pluripotency guarding adhesions

Human pluripotent stem cells (hPSC) can generate almost all adult cell lineages. While it is clear that key transcriptional programmes are important elements for maintaining pluripotency, the equally essential requirement for cell adhesion to specific extracellular matrix components remains poorly defined. Our recent observation that hPSC colonies form unusually large \"cornerstone\" focal adhesions (FA), distinct from parental somatic cells, that are lost following differentiation, emphasises the potential of these atypical FA as gatekeepers of pluripotency. Here, using nanopatterns, we further demonstrate that physical restriction of adhesion size, in hPSC colonies, is sufficient to trigger differentiation. Using superresolution two-colour interfero-metric photo-activated localization microscopy (iPALM), we examined the three-dimensional architecture of these cornerstone adhesions and report vertical lamination of FA proteins with three main structural peculiarities: 1) integrin {beta}5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall. 2) Vinculin localises higher than expected with respect to the substrata and displays a head-above-tail orientation, and 3) surprisingly, actin and -actinin are present in two discrete layers, a previously undescribed localisation for these proteins. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a key role for kanks in hPSC colony architecture.

cell biology↗

bioRxiv2018

Filopodome mapping identifies p130Cas as a mechanosensitive regulator of filopodia stability

Filopodia are adhesive cellular protrusions specialised in the detection of extracellular matrix (ECM)-derived cues. While ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins (\"adhesome\") to fine-tune cellular behaviour, the components of the filopodia adhesions remain undefined. Here, we performed a structured illumination microscopy-based screen to map the localisation of 80 target proteins, linked to cell adhesion and migration, within filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phospho-inositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localisation of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. Finally, we demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its CCHD domain and acts as a mechanosensitive regulator of filopodia stability.

cell biology↗

bioRxiv2018

GGA2 and RAB13 regulate activity-dependent β1-integrin recycling

{beta}1-integrins mediate cell-matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes like cancer cell invasion. Here we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with {beta}1-integrin and its role in recycling of the active but not inactive {beta}1-integrin receptors. Silencing of GGA2 limits active {beta}1-integrin levels in focal adhesions and decreases cancer cell migration and invasion congruent with its ability to regulate the dynamics of active integrins. Using the proximity-dependent biotin identification (BioID) method, we identify two RAB family small GTPases, RAB13 and RAB10, associating with GGA2 and {beta}1-integrin. Functionally, RAB13 silencing triggers the intracellular accumulation of active {beta}1-integrin, reduces integrin activity, in focal adhesions, and cell migration, similarly to GGA2 depletion, indicating that both facilitate active {beta}1-integrin recycling the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.

cell biology↗

bioRxiv2018

SORLA-driven endosomal trafficking regulates the oncogenic fitness of HER2

Human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. Endosomal trafficking of many other receptor tyrosine kinases regulates their oncogenic signaling, but the prevailing view is that HER2 is retained on the cell surface. Here we reveal that in cancer cells Sortilin related receptor 1 (SORLA; SORL1) forms a complex with HER2 and regulates its subcellular distribution by promoting recycling of endosomal HER2 back to plasma membrane. Expression of SORLA in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA targets HER2 to late endosomal/lysosomal compartments, impairs HER2-driven signaling and in vivo tumor growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

cell biology↗

bioRxiv2017

ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno-isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Iinenteractions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual or {beta} subunits or as {beta} heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.

molecular biology↗

bioRxiv2017

Vimentin Plays A Functional Role In Mammary Gland Regeneration

In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and in basal epithelial cell populations including gland-reconstituting mammary stem cells (MaSC), with largely undefined functions. Here, we studied how vimentin deficiency affects mouse mammary gland development. Our results demonstrate that in adult vimentin knockout mice (Vim-/-) mammary ductal outgrowth is delayed. The adult Vim-/- glands are characterised by dilated ducts, an imbalance in the proportion of basal to luminal mammary epithelial cells and a reduction in cells expressing Slug (Snai2), an established MaSC regulator. All of these features are indicative of reduced progenitor cell activity. Accordingly, isolated Vim-/- mammary epithelial cells display reduced capacity to form mammospheres, and altered organoid structure, compared to wt counterparts, when plated in a 3D matrix in vitro. Importantly, altered basal epithelial cell number translates into defects in Vim-/- mammary gland regeneration in vivo in cleared fat pad transplantation studies. Furthermore, we show that vimentin contributes to stem-like cell properties in triple negative MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces tumorsphere formation and alters expression of breast cancer stem cell-associated surface markers. Together, our findings identify vimentin as a positive regulator of stemness in the developing mouse mammary gland and in breast cancer cells.

developmental biology↗

bioRxiv2017

FiloQuant reveals increased filopodia density during DCIS progression

Filopodia are commonly observed cellular protrusions in vitro and in vivo. Defective filopodia formation is linked to several pathologies including cancer, wherein actively protruding filopodia, at the invasive front, and filopodia-mediated probing of the microenvironment accompanies cancer cell dissemination. Despite wide biological significance, delineating the function of these finger-like protrusions in more complex systems remains technically challenging, particularly hindered by lack of compatible methods to quantify filopodia properties. Here, we present FiloQuant, a freely available ImageJ plugin, to detect filopodia and filopodia-like protrusions in both fixed and live-cell microscopy data. We demonstrate that FiloQuant can extract quantifiable information including protrusion dynamics, density and length from multiple cell types and in a range of microenvironments, such as during collective or single cancer cell migration in 2D and 3D, in fixed neuronal cultures, in activated natural killer cells and in sprouting endothelial cells in vivo. In cellular models of breast ductal carcinoma in situ (DCIS) we reveal a link between filopodia formation at the cell-matrix interface, during collective invasion and in 3D tumour spheroids, with the previously reported local invasive potential of these breast cancer models in vivo. Finally, using intravital microscopy, we observed that tumour spheroids display prominent filopodia in vivo, supporting a potential role for these protrusions during tumorigenesis.

cell biology↗