Abstract:Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting non-genomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5, and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared to cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn upregulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a novel non-genomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity and cell migratory capacity. 3 Significance:Cells require nuclear deformation to squeeze through tissue matrices. We have discovered that WDR5 (an epigenetic modulator of H3K4 methylation) is fundamental for cell polarity and migration in vitro and in vivo, independently of transcription. We have uncovered that the interactions between cells and the surrounding 3D confined conditions induce the upregulation of H3K4me3. Moreover, 3D environments control the deformability and the mechanical properties of the nucleus. We have identified that loss of WDR5 abrogates the H3K4 methylation and the nuclear changes induced by 3D conditions. Mechanistically, we found that MLCK and myosin function was required for WDR5-mediated H3K4 methylation in 3D matrices. Our findings uncover new functions of the epigenetic machinery when cells move through constricted conditions.

Cancer cell migration is a complex process that requires coordinated structural changes and signals in multiple cellular compartments. The nucleus is the biggest and stiffest organelle of the cell and might alter its physical properties to allow cancer cell movement. Integrins are transmembrane receptors that mediate cell-cell and cell-extracellular matrix interactions, which regulate numerous intracellular signals and biological functions under physiological conditions. Moreover, integrins orchestrate changes in tumor cells and their microenvironment that lead to cancer growth, survival and invasiveness. Most of the research efforts have focused on targeting integrin-mediated adhesion and signaling. Recent exciting data suggest the crucial role of integrins in controlling internal cellular structures and nuclear alterations during cancer cell migration. Here we review the emerging role of integrins in nuclear biology. We highlight increasing evidence that integrins are critical for changes in multiple nuclear components, the positioning of the nucleus and its mechanical properties during cancer cell migration. Finally, we discuss how integrins are integral proteins linking the plasma membrane and the nucleus, and how they control cell migration to enable cancer invasion and infiltration. The functional connections between these cell receptors and the nucleus will serve to define new attractive therapeutic targets.

The nucleus is fundamentally composed by lamina and nuclear membranes that enclose the chromatin, nucleoskeletal components and suspending nucleoplasm. the functional connections of this network integrate external stimuli into cell signals, including physical forces to mechanical responses of the nucleus. Canonically, the morphological characteristics of the nucleus, as shape and size, have served for pathologists to stratify and diagnose cancer patients; however, novel biophysical techniques must exploit physical parameters to improve cancer diagnosis. By using multiple particle tracking (Mpt) technique on chromatin granules, we designed a SURF (Speeded Up Robust Features)-based algorithm to study the mechanical properties of isolated nuclei and in living cells. We have determined the apparent shear stiffness, viscosity and optical density of the nucleus, and how the chromatin structure influences on these biophysical values. Moreover, we used our MPT-SURF analysis to study the apparent mechanical properties of isolated nuclei from patients of acute lymphoblastic leukemia. We found that leukemia cells exhibited mechanical differences compared to normal lymphocytes. Interestingly, isolated nuclei from high-risk leukemia cells showed increased viscosity than their counterparts from normal lymphocytes, whilst nuclei from relapsed-patient's cells presented higher density than those from normal lymphocytes or standard-and high-risk leukemia cells. Taken together, here we presented how MPT-SURF analysis of nuclear chromatin granules defines nuclear mechanical phenotypic features, which might be clinically relevant. The nucleus is a central cellular organelle that must alter its physical properties during cellular functions, including gene expression, cell migration, and development in homeostasis and human diseases 1. The nucleus is composed by the nuclear envelope, nucleoskeletal components, and the nucleoplasm, which contains the DNA and its associated molecules forming the chromatin 2. The nuclear envelope is mainly composed by nuclear membranes, A-(lamin A and C) and B-(lamin B) lamin types, and other structural proteins that connect the nucleus with the cytoskeleton as LINC complexes 3. Lamin A/C levels and its ratio to lamin B levels control nuclear deformability and stiffness 4,5. It has been reported that other nuclear components, as LINC and F-actin binding proteins, control nuclear shape and rigidity 6. In general, these nuclear changes correlate with more invasive phenotype of tumor cells and higher genomic instability upon cell migration 7,8. Chromatin organization is modulated by epigenetic changes that promote chromatin compaction and decondensation according to electrostatic interactions and configurational entropy 9-11. Several biophysical techniques support that the chromatin conformation alterations contributes to the morphology and the biophysical behavior of the nucleus 12-16 Abnormalities in nuclear shape and organization occur in a wide range of human pathologies,