Culturing and Imaging Glioma Stem Cells in 3D Collagen Matrices.

Methods to maintain human glioma stem cells as neurosphere cultures and image their dynamic behavior in 3D collagen matrices are described. Additional approaches to monitor glioma stem cell differentiation into mesenchymal-type cells, along with example data are included. Together, these approaches enable glioma stem cell differentiation to be controlled while maintaining the cells in culture, as well as allowing cell dynamics to be captured and analyzed. These methods should be helpful for those seeking to understand the molecular mechanisms driving the invasion of glioma cells through three-dimensional environments. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Culturing human glioma stem cells as neurospheres Basic Protocol 2: Inducing GSC adherence and monitoring their differentiation into mesenchymal cells Support Protocol 1: Preparing fibronectin-coated dishes for cell microscopy Basic Protocol 3: Embedding GSCs in a 3D collagen matrix to study their invasive behavior Support Protocol 2: Phase-contrast imaging with a tiled matrix to study cell migration in a 3D gel.

Non-muscle myosin II and the plasticity of 3D cell migration.

Confined cells migrating through 3D environments are also constrained by the laws of physics, meaning for every action there must be an equal and opposite reaction for cells to achieve motion. Fascinatingly, there are several distinct molecular mechanisms that cells can use to move, and this is reflected in the diverse ways non-muscle myosin II (NMII) can generate the mechanical forces necessary to sustain 3D cell migration. This review summarizes the unique modes of 3D migration, as well as how NMII activity is regulated and localized within each of these different modes. In addition, we highlight tropomyosins and septins as two protein families that likely have more secrets to reveal about how NMII activity is governed during 3D cell migration. Together, this information suggests that investigating the mechanisms controlling NMII activity will be helpful in understanding how a single cell transitions between distinct modes of 3D migration in response to the physical environment.

Cytoplasmic pressure maintains epithelial integrity and inhibits cell motility.

Cytoplasmic pressure, a function of actomyosin contractility and water flow, can regulate cellular morphology and dynamics. In mesenchymal cells, cytoplasmic pressure powers cell protrusion through physiological three-dimensional extracellular matrices. However, the role of intracellular pressure in epithelial cells is relatively unclear. Here we find that high cytoplasmic pressure is necessary to maintain barrier function, one of the hallmarks of epithelial homeostasis. Further, our data show that decreased cytoplasmic pressure facilitates lamellipodia formation during the epithelial to mesenchymal transition (EMT). Critically, activation of the actin nucleating protein Arp2/3 is required for the reduction in cytoplasmic pressure and lamellipodia formation in response to treatment with hepatocyte growth factor (HGF) to induce EMT. Thus, elevated cytoplasmic pressure functions to maintain epithelial tissue integrity, while reduced cytoplasmic pressure triggers lamellipodia formation and motility during HGF-dependent EMT.

The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes.

MYO19 interacts with mitochondria through a C-terminal membrane association domain (MyMOMA). Specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using promiscuous biotinylation data in combination with existing affinity-capture databases, we have identified a number of putative MYO19-interacting proteins. We chose to explore the interaction between MYO19 and the mitochondrial GTPase Miro2 by expressing mchr-Miro2 in combination with GFP-tagged fragments of the MyMOMA domain and assaying for recruitment of MYO19-GFP to mitochondria. Coexpression of MYO19898-970 -GFP with mchr-Miro2 enhanced MYO19898-970 -GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19898-970 -GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization-activated reduction in fluorescence analysis. MyMOMA constructs containing a putative membrane-insertion motif but lacking the Miro2-interacting region displayed slow exchange kinetics. MYO19898-970 -GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that MYO19 interacting with Miro2 has higher mobility than MYO19 inserted into the mitochondrial outer membrane. Mutation of well-conserved, charged residues within MYO19 or within the switch I and II regions of Miro2 abolished the enhancement of MYO19898-970 -GFP localization in cells ectopically expressing mchr-Miro2. Additionally, expressing mutant versions of Miro2 thought to represent particular nucleotide states indicated that the enhancement of MYO19898-970 -GFP localization is dependent on Miro2 nucleotide state. Taken together, these data suggest that membrane-inserted MYO19 is part of a larger complex, and that Miro2 plays a role in integration of actin- and microtubule-based mitochondrial activities.