Search

We present here a novel multi-parametric approach for the characterization of multiple cellular features, using images acquired by high-throughput and high-definition light microscopy. We specifically used this approach for deep and unbiased analysis of the effects of a drug library on five cultured cell lines. The presented method enables the acquisition and analysis of millions of images, of treated and control cells, followed by an automated identification of drugs inducing strong responses, evaluating the median effect concentrations and those cellular properties that are most highly affected by the drug. The tools described here provide standardized quantification of multiple attributes for systems level dissection of complex functions in normal and diseased cells, using multiple perturbations. Such analysis of cells, derived from pathological samples, may help in the diagnosis and follow-up of treatment in patients.

We present here a novel multi-parametric approach for the characterization of multiple cellular features, using images acquired by high-throughput and high-definition light microscopy. We specifically used this approach for deep and unbiased analysis of the effects of a drug library on five cultured cell lines. The presented method enables the acquisition and analysis of millions of images, of treated and control cells, followed by an automated identification of drugs inducing strong responses, evaluating the median effect concentrations and those cellular properties that are most highly affected by the drug. The tools described here provide standardized quantification of multiple attributes for systems level dissection of complex functions in normal and diseased cells, using multiple perturbations. Such analysis of cells, derived from pathological samples, may help in the diagnosis and follow-up of treatment in patients.

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.