Genome-wide Maps of Nuclear Lamina Interactions in Single Human Cells

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization.National Institutes of Health (U.S.) (Grant R01 GM114190)National Human Genome Research Institute (U.S.) (Grant R01 HG003143

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion

Modular actin nano-architecture enables podosome protrusion and mechanosensing

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries

Unraveling Intermediate Filaments: The super resolution solution

Intermediate Filaments (IFs) carry out major functions in cells. Several diseases have been associated with malfunctioning IFs in the cells and among them are certain sub types of cancer. To determine the structure and organization of IFs, we have used Single Molecule Localization Microscopy (SMLM) . We have shown that keratin IF plays a key role in Hemidesmosomes (HDs) organization in cultured keratinocytes. In addition, we have studied the orientational alignment between vimentin, another abundant IF protein, and MT. With SR, we have observed spatial proximity between single vimentin and MT filaments and we have quantitatively shown that interaction between vimentin and MT are cell-type dependent. Furthermore, we have addressed several critical issues in SMLM imaging. First, we have developed a new buffer which supports multi-color imaging. OxEA, an Oxyrase based imaging buffer, elevates localization precision for commonly used dyes and enhances blinking of other dyes. Second we have improved a dedicated chamber for SR imaging, so called Oxygen Tight Chamber (OTC). In the OTC, we have minimized drift by introducing an alternative curing procedure that reduces stress in the bond between glass and plastic. In brief, in this thesis we have optimized imaging condition and post processing analysis for SMLM microscopy. With the resulting high quality SR images and precise quantification methods, we have contributed to the biological knowledge in the IF field

Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway

Autophagy is the main homeostatic pathway guiding cytosolic materials for degradation by the lysosome. Maturation of autophagosomes requires their transport towards the perinuclear region of the cell, with key factors underlying both processes still poorly understood. Here we show that transport and positioning of late autophagosomes depends on cholesterol by way of the cholesterol-sensing Rab7 effector ORP1L. ORP1L localizes to late autophagosomes and—under low-cholesterol conditions—contacts the ER protein VAP-A, forming ER-autophagosome contact sites, which prevent minus-end transport by the Rab7–RILP–dynein complex. ORP1L-mediated contact sites also inhibit localization of PLEKHM1 to Rab7. PLEKHM1, together with RILP, then recruits the homotypic fusion and vacuole protein-sorting (HOPS) complex for fusion of autophagosomes with late endosomes and lysosomes. Thus, ORP1L, via its liganding by lipids and the formation of contacts between autophagic vacuoles and the ER, governs the last steps in autophagy that lead to the lysosomal degradation of cytosolic material

Hemidesmosomes modulate force generation via focal adhesions.

Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6β4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6β4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6β4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho-ROCK-MLC- and FAK-PI3K-dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVβ5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes

Hemidesmosomes modulate force generation via focal adhesions

Biological and Soft Matter Physic