Kristiana Kandere-Grzybowska

Molecular dynamics imaging in micropatterned living cells.

Micropatterning approaches using self-assembled monolayers of alkyl thiols on gold are not optimal for important imaging modalities in cell biology because of absorption of light and scattering of electrons by the gold layer. We report here an anisotropic solid microetching (ASOMIC) procedure that overcomes these limitations. The method allows molecular dynamics imaging by wide-field and total internal reflection fluorescence (TIRF) microscopy of living mammalian cells and correlative platinum replica electron microscopy.

Cell motility on micropatterned treadmills and tracks

Surfaces micropatterned with disjointed cell adhesive/non-adhesive regions allow for precise control of cell shape, internal organization and function. In particular, substrates prepared by the reaction-diffusion ASoMic (nisotropic lid roetching) method localize cells onto transparent micro-islands or tracks surrounded by an opaque, adhesion-resistant background. ASoMic is compatible with several important imaging modalities ( wide-field, fluorescent, TIRF and confocal microscopies), and can be used to study and quantify various intracellular and cellular processes related to cell motility. For cells constrained on the islands, the imposed geometry controls spatial organization of the cytoskeleton, while the transparency of the islands allows for real-time analysis of cytoskeletal dynamics. For cells on transparent, linear tracks, the high optical contrast between these adhesive regions and the surrounding non-adhesive background allows for straightforward quantification of the key parameters describing cell motility. Both types of systems provide analytical-quality data that can assist fundamental studies of cell locomotion and can provide a technological basis for cell motility microassays.

Microtubule guidance tested through controlled cell geometry

Summary In moving cells dynamic microtubules (MTs) target and disassemble substrate adhesion sites (focal adhesions; FAs) in a process that enables the cell to detach from the substrate and propel itself forward. The short-range interactions between FAs and MT plus ends have been observed in several experimental systems, but the spatial overlap of these structures within the cell has precluded analysis of the putative long-range mechanisms by which MTs growing through the cell body reach FAs in the periphery of the cell. In the work described here cell geometry was controlled to remove the spatial overlap of cellular structures thus allowing for unambiguous observation of MT guidance. Specifically, micropatterning of living cells was combined with high-resolution in-cell imaging and gene product depletion by means of RNA interference to study the long-range MT guidance in quantitative detail. Cells were confined on adhesive triangular microislands that determined cell shape and ensured that FAs localized exclusively at the vertices of the triangular cells. It is shown that initial MT nucleation at the centrosome is random in direction, while the alignment of MT trajectories with the targets (i.e. FAs at vertices) increases with an increasing distance from the centrosome, indicating that MT growth is a non-random, guided process. The guided MT growth is dependent on the presence of FAs at the vertices. The depletion of either myosin IIA or myosin IIB results in depletion of F-actin bundles and spatially unguided MT growth. Taken together our findings provide quantitative evidence of a role for long-range MT guidance in MT targeting of FAs.

Carboxybetaine Methacrylate Polymers Offer Robust, Long-Term Protection against Cell Adhesion

Films of poly(carboxybetaine methacrylate), poly(CBMA), grafted onto microetched gold slides are effective in preventing nonspecific adhesion of cells of different types. The degree of adhesion resistance is comparable to that achieved with the self-assembled monolayers, SAMs, of oligo(ethylene glycol) alkanethiolates. In sharp contrast to the SAMs, however, substrates protected with poly(CBMA) can be stored in dry state without losing their protective properties for periods up to 2 weeks.

Why Cells are Microscopic: A Transport-Time Perspective

Physical-chemical reasoning is used to demonstrate that the sizes of both prokaryotic and eukaryotic cells are such that they minimize the times needed for the macromolecules to migrate throughout the cells and interact/react with one another. This conclusion does not depend on a particular form of the crowded-medium diffusion model, as thus points toward a potential optimization principle of cellular organisms. In eukaryotes, size optimality renders the diffusive transport as efficient as active transport - in this way, the cells can conserve energetic resources that would otherwise be expended in active transport.

Tomography and Static-Mechanical Properties of Adherent Cells

A tomography approach is used to reconstruct 3D cell shapes and, simultaneously, the shapes/positions of the nuclei within these cells. Subjecting the cells to well-defined microconfinements of various diameters allow for relating the steady-state shapes of cells to their static-mechanical properties. The observed shapes show striking regularities between different cell types and all fit to a model that takes into account the cell membrane, cortical actin, and the nucleus.

Nanoparticle-Based Solution Deposition of Gold Films Supporting Bioresistant SAMs

Thin films of gold on glass are prepared by solution deposition of functionalized gold nanoparticles followed by thermal treatment. The processed films adhere strongly to glass without any adhesion layers and can be micropatterned/microetched without delamination from the substrate. The formation of self-assembled monolayers (SAMs) of oligo(ethylene glycol) alkane thiols (EG SAMs) renders the films resistant to cell adhesion and allows for cell patterning.

Lévy-like movement patterns of metastatic cancer cells revealed in microfabricated systems and implicated in vivo

Metastatic cancer cells differ from their non-metastatic counterparts not only in terms of molecular composition and genetics, but also by the very strategy they employ for locomotion. Here, we analyzed large-scale statistics for cells migrating on linear microtracks to show that metastatic cancer cells follow a qualitatively different movement strategy than their non-invasive counterparts. The trajectories of metastatic cells display clusters of small steps that are interspersed with long “flights”. Such movements are characterized by heavy-tailed, truncated power law distributions of persistence times and are consistent with the Lévy walks that are also often employed by animal predators searching for scarce prey or food sources. In contrast, non-metastatic cancerous cells perform simple diffusive movements. These findings are supported by preliminary experiments with cancer cells migrating away from primary tumors in vivo. The use of chemical inhibitors targeting actin-binding proteins allows for “reprogramming” the Lévy walks into either diffusive or ballistic movements.