Modes of remodeling in the cortical cytoskeleton of vascular endothelial cells

AbstractThe cortical cytoskeleton of vascular endothelial cells plays an important role in responding to mechanical stimuli and controlling the distribution of cell surface proteins. Here, we have used atomic force microscopy to visualize the dynamics of cortical cytoskeleton in living bovine pulmonary artery endothelial cells. We demonstrate that the cortical cytoskeleton, organized as a complex polygonal mesh, is highly dynamic and shows two modes of remodeling: intact-boundary-mode where mesh element boundaries remain intact but move at ∼0.08μm/min allowing the mesh element to change shape, and altered-boundary-mode where new mesh boundaries form and existing ones disappear

Fabrication of 3D Controlled in vitro Microenvironments

Microfluidics-based lab-on-a-chips have many advantages, one of which is to provide physiologically relevant settings for cell biology experiments. Thus there is an ever increasing interest in their fabrication. Our goal is to construct three dimensional (3D) Controlled in vitro Microenvironments (CivMs) that mimic the in vivo microenvironments. Here, we present our optimized fabrication method that works for various lab-on-a-chip designs with a wide range of dimensions. The most crucial points are:While using one type of SU-8 photoresist (SU-2075), fine tuning of ramp, dwell time, spin speed, durations of soft bake, UV exposure and development allows fabrication of SU-8 masters with various heights from 40 to 600 μm.Molding PDMS (polydimethylsiloxane) at room temperature for at least two days instead of baking at higher temperatures prevents not only tears and bubbles in PDMS stamps but also cracks in the SU-8 master.3D nature of the CivMs is ensured by keeping the devices inverted during gel polymerization

Invadopodia: Proteolytic feet of cancer cells

The leading cause of death in cancer patients is metastasis. Invasion is an integral part of metastasis and is carried out by proteolytic structures called invadopodia at the cellular level. In this introductory review, we start by evaluating the definition of invadopodia. While presenting the upstream signaling events involved, we integrate current models on invadopodia. In addition, we discuss the significance of invadopodia in 2D and 3D and in vivo. We finally point out technical challenges and conclude with open questions in the field. © TÜBİTAK.Scientific and Technological Research Council of Turkey (111T547

Micrometer scale spacings between fibronectin nanodots regulate cell morphology and focal adhesions

Cell adhesion to extracellular matrix is an important process for both health and disease states. Surface protein patterns that are topographically flat, and do not introduce other chemical, topographical or rigidity related functionality and, more importantly, that mimic the organization of the in vivo extracellular matrix are desired. Previous work showed that vinculin and cytoskeletal organization are modulated by size and shape of surface nanopatterns. However, quantitative analysis on cell morphology and focal adhesions as a function of micrometer scale spacings of FN nanopatterns was absent. Here, electron beam lithography was used to pattern fibronectin nanodots with micrometer scale spacings on a Kcasein background on indium tin oxide coated glass which, unlike silicon, is transparent and thus suitable for many light microscopy techniques. Exposure times were significantly reduced using the line exposure mode with micrometer scale step sizes. Micrometer scale spacings of 2, 4 and 8 m between fibronectin nanodots proved to modulate cell adhesion through modification of cell area, focal adhesion number, size and circularity. Overall, cell behavior was shown to shift at the apparent threshold of 4 m spacing. The findings presented here offer exciting new opportunities for cell biology research.TUBITAK (111T026); DPT (2009K120860

Step-by-step quantitative analysis of focal adhesions

Focal adhesions (FAs) are specialized adhesive structures which serve as cellular communication units between cells and the surrounding extracellular matrix. FAs are involved in signal transduction and actin cytoskeleton organization. FAs mediate cell adhesion, which is a critical phenomenon in cancer research. Since cells can form many and micrometer scale FAs, their quantitative analysis demands well-optimized image analysis approaches [1-3]. Here, we have optimized the analysis of FAs of MDA-MB-231 breast cancer cells. The optimization is based on proper processing of immunofluorescence images of vinculin, which is one of the markers of FAs. All image processing steps are carried out using the ImageJ software, which is freely available and in the public domain. The advantages of our method are:The analysis steps are simplified by combining different plugins of the ImageJ program.FAs are better detected with minimal false negatives due to optimized processing of fluorescent images.This approach can be applied to quantify a variety of fluorescent images comprising focal and/or localized signals within a high background such as FAs, one of the many complex signaling structures in a cell

Cellular distribution of invadopodia is regulated by nanometer scale surface protein patterns

Invadopodia are proteolytic structures formed by cancer cells. It is not known whether their cellular distribution can be regulated by the organization of the extracellular matrix or the organization of the golgi complex or whether they have an adhesion requirement. Here, we used electron beam lithography to fabricate fibronectin (FN) nanodots with isotropic and gradient micrometer scale spacings on K-casein and laminin backgrounds. Investigating cancer cells cultured on protein nanopatterns, we showed that (i) presence of FN nanodots on a K-casein background decreased percent of cells with neutral invadopodia polarization compared to FN control surfaces; (ii) presence of a gradient of FN nanodots on a K-casein background increased percent of cells with negative invadopodia polarization compared to FN control surfaces; (iii) polarization of the golgi complex was similar to that of invadopodia in agreement with a spatial link; (iv) local adhesion did not necessarily appear to be a prerequisite for invadopodia formation.Scientific and Technological Research Council of Turkey (TUBITAK 111T547) State Planning Organization (DPT 2009K120860

Automated segmentation of cells in phase contrast optical microscopy time series images

2019 Medical Technologies Congress, TIPTEKNO 2019; Palm Wings Ephesus HotelIzmir; Turkey; 3 October 2019 through 5 October 2019Phase contrast optical microscopy is a preferred imaging technique for live-cell, temporal analysis. Segmentation of cells from time series data acquired with this technique is a labor-intensive and time-consuming task that cell biology researchers need solution for. In this study traditional image processing and deep learning based approaches for automated cell segmentation from phase contrast optical microscopy time series are presented, and their performances are evaluated against manually annotated datasets. © 2019 IEEE.Faz kontrast optik mikroskopi hücrelerin canlı ortamlarında zamana bağlı incelenmesi için tercih edilen görüntüleme yöntemidir. Bu yöntem ile elde edilen zaman serisi görüntülerinde hücrelerin bölütlenmesi işi hücre biyolojisi araştırmacılarının çözümüne ihtiyaç duyduğu emek yoğun ve zaman alan bir iştir. Bu çalışmada faz kontrast optik mikroskopi zaman serilerinde hücrelerin otomatik bölütlenmesi için geleneksel görüntü işleme ve derin öğrenme temelli yöntemler önerilmiş ve başarımları elle işaretlenmiş veri kümelerinde nicel olarak ölçülmüştür

Breast cancer cells and macrophages in a paracrine-juxtacrine loop

Breast cancer cells (BCC) and macrophages are known to interact via epidermal growth factor (EGF) produced by macrophages and colony stimulating factor-1 (CSF-1) produced by BCC. Despite contradictory findings, this interaction is perceived as a paracrine loop. Further, the underlying mechanism of interaction remains unclear. Here, we investigated interactions of BCC with macrophages in 2D and 3D. While both BCC and macrophages showed invasion/chemotaxis to fetal bovine serum, only macrophages showed chemotaxis to BCC in custom designed 3D cell-on-a-chip devices. These results were in agreement with gradient simulation results and ELISA results showing that macrophage-derived-EGF was not secreted into macrophage-conditioned-medium. Live cell imaging of BCC in the presence and absence of iressa showed that macrophages but not macrophage-derived matrix modulated adhesion and motility of BCC in 2D. 3D co-culture experiments in collagen and matrigel showed that BCC changed their multicellular organization in the presence of macrophages. In custom designed 3D co-culture cell-on-a-chip devices, macrophages promoted and reduced migration of BCC in collagen and matrigel, respectively. Furthermore, adherent but not suspended BCC endocytosed EGFR when in contact with macrophages. Collectively, our data revealed that macrophages showed chemotaxis towards BCC whereas BCC required direct contact to interact with macrophage-derived-EGF. Therefore, we propose that the interaction between cancer cells and macrophages is a paracrine-juxtacrine loop of CSF-1 and EGF, respectively

Evidence for a Highly Elastic Shell-Core Organization of Cochlear Outer Hair Cells by Local Membrane Indentation

Cochlear outer hair cells (OHCs) are thought to play an essential role in the high sensitivity and sharp frequency selectivity of the hearing organ by generating forces that amplify the vibrations of this organ at frequencies up to several tens of kHz. This tuning process depends on the mechanical properties of the cochlear partition, which OHC activity has been proposed to modulate on a cycle-by-cycle basis. OHCs have a specialized shell-core ultrastructure believed to be important for the mechanics of these cells and for their unique electromotility properties. Here we use atomic force microscopy to investigate the mechanical properties of isolated living OHCs and to show that indentation mechanics of their membrane is consistent with a shell-core organization. Indentations of OHCs are also found to be highly nonhysteretic at deformation rates of more than 40 μm/s, which suggests the OHC lateral wall is a highly elastic structure, with little viscous dissipation, as would appear to be required in view of the very rapid changes in shape and mechanics OHCs are believed to undergo in vivo