Julien Colombelli

Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization

The mechanics of the actin cytoskeleton have a central role in the regulation of cells and tissues, but the details of how molecular sensors recognize deformations and forces are elusive. By performing cytoskeleton laser nanosurgery in cultured epithelial cells and fibroblasts, we show that the retraction of stress fibers (SFs) is restricted to the proximity of the cut and that new adhesions form at the retracting end. This suggests that SFs are attached to the substrate. A new computational model for SFs confirms this hypothesis and predicts the distribution and propagation of contractile forces along the SF. We then analyzed the dynamics of zyxin, a focal adhesion protein present in SFs. Fluorescent redistribution after laser nanosurgery and drug treatment shows a high correlation between the experimentally measured localization of zyxin and the computed localization of forces along SFs. Correlative electron microscopy reveals that zyxin is recruited very fast to intermediate substrate anchor points that are highly tensed upon SF release. A similar acute localization response is found if SFs are mechanically perturbed with the cantilever of an atomic force microscope. If actin bundles are cut by nanosurgery in living Drosophila egg chambers, we also find that zyxin redistribution dynamics correlate to force propagation and that zyxin relocates at tensed SF anchor points, demonstrating that these processes also occur in living organisms. In summary, our quantitative analysis shows that force and protein localization are closely correlated in stress fibers, suggesting a very direct force-sensing mechanism along actin bundles.

In vivo Selective Cytoskeleton Dynamics Quantification in Interphase Cells Induced by Pulsed Ultraviolet Laser Nanosurgery

We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage‐rate measurements in interphase Ptk‐2 cells throughout the entire cell. We quantify the impact of two labeling methods and three fluorescent markers, showing a 25% faster depolymerization with Alexa‐488 tubulin compared with Rhodamine and yellow fluorescent protein (YFP) tubulins and a 20% higher variability induced by microinjection compared with stable transfection. Using EB3‐GFP as a tip marker, we establish a new protocol to measure shrinkage rate, growth rate and rescue frequency simultaneously with high temporal and spatial specificity in live cells. As our analysis shows, laser‐induced MT dynamics are physiologically relevant. The high statistical efficiency that the method offers in terms of numbers of measured events and therefore reduced standard deviations represents an important quantitative improvement in the measurement of dynamic instability parameters in vivo. We extend the application of the method by demonstrating induced dynamic behavior of actin‐stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement.

Ultraviolet diffraction limited nanosurgery of live biological tissues

A laser nanodissection system for in vivo and in situ biological tissues is presented. A pulsed laser beam operating at a wavelength of 355 nm enables diffraction limited dissection, providing an optimal tool for intracellular nanosurgery. Coupled into a conventional inverted microscope and scanned across a field of up to 100×100 μm2, this optical nanoscalpel performs in vivo photoablation and plasma-induced ablation inside organisms ranging from intracellular organelles to embryos. The system allows the use of conventional microscopy contrasts and methods, fast dissection with up to 1000 shots per second, and simultaneous dissection and imaging. This article outlines an efficient implementation with a small number of components and reports an improvement of this state of the art of plasma-induced ablation technique over previous studies, with a ratio of plasma volume to beam focal volume of 5.2. This offers, e.g., the possibility of writing information directly at the sample location by plasma glass nano...

Spore number control and breeding in Saccharomyces cerevisiae: a key role for a self-organizing system

Spindle pole bodies (SPBs) provide a structural basis for genome inheritance and spore formation during meiosis in yeast. Upon carbon source limitation during sporulation, the number of haploid spores formed per cell is reduced. We show that precise spore number control (SNC) fulfills two functions. SNC maximizes the production of spores (1–4) that are formed by a single cell. This is regulated by the concentration of three structural meiotic SPB components, which is dependent on available amounts of carbon source. Using experiments and computer simulation, we show that the molecular mechanism relies on a self-organizing system, which is able to generate particular patterns (different numbers of spores) in dependency on one single stimulus (gradually increasing amounts of SPB constituents). We also show that SNC enhances intratetrad mating, whereby maximal amounts of germinated spores are able to return to a diploid lifestyle without intermediary mitotic division. This is beneficial for the immediate fitness of the population of postmeiotic cells.

Reprogramming cell shape with laser nano-patterning

Cell shape in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the cell microenvironment. Here, we manipulate the shape of living cells in real time by using a tightly focused pulsed laser to introduce additional geometrically defined adhesion sites. The sub-micrometer resolution of the laser patterning allowed us to identify the critical distances between cell adhesion sites required for cell shape extension and contraction. This easy-to-handle method allows the precise control of specific actin-based structures that regulate cell architecture. Actin filament bundles or branched meshworks were induced, displaced or removed in response to specific dynamic modifications of the cell adhesion pattern. Isotropic branched actin meshworks could be forced to assemble new stress fibers locally and polarised in response to specific geometrical cues.

Dynein‐mediated pulling forces drive rapid mitotic spindle elongation in Ustilago maydis

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein‐mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin‐5, which is located in the spindle mid‐zone. When the spindle reaches ∼2 μm in length, the elongation rate increases four‐fold. This coincides with the appearance of long and less‐dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser‐mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less‐dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off‐loaded from the MT plus‐end to the cortex.

Three-dimensional laser microsurgery in light-sheet based microscopy (SPIM)

Advances in the life sciences rely on the ability to observe dynamic processes in live systems and in environments that mimic in-vivo situations. Therefore, new methodological developments have to provide environments that resemble physiologically and clinically relevant conditions as closely as possible. In this work, plasma-induced laser nanosurgery for three-dimensional sample manipulation and sample perturbation is combined with optically sectioning light-sheet based fluorescence microscopy (SPIM) and applied to three-dimensional biological model systems. This means: a) working with a biological system that is not confined to essentially two dimensions like cell cultures on cover glasses, b) gaining intrinsic optical sectioning capabilities by an efficient three-dimensional fluorescence imaging system, and c) using arbitrarily-shaped three-dimensional ablation-patterns by a plasma-induced laser ablation system that prevent damage to surrounding tissues. Spatial levels in our biological applications range from sub-microns during delicate ablation of single microtubules over the confined disruption of cell membranes in an MDCK-cyst to the macroscopic cutting of a millimeter-sized Zebrafish caudal fin with arbitrary three-dimensional ablation patterns. Dynamic processes like laser-induced hemocyte migration can be studied with our SPIM-microscalpel in intact, live embryos.

Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage

For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1Cdt2 ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21Cip1 also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21Cip1 exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation.

Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer’s disease mouse models

Loss of GPR3 reduced amyloid plaque burden and improved cognition in four mouse models of Alzheimer’s disease, suggesting that GPR3 may be a potential therapeutic target. GPR3, a therapeutic target for AD? Alzheimer’s disease (AD) is characterized by the degeneration of brain networks involved in cognitive function. AD mouse models are used to study disease pathogenesis, but no single model fully captures the pathological changes in AD patients. Thus, extensive validation of AD therapeutic targets in multiple animal models is required before advancing to clinical research. In new work, Huang et al. determined that the absence of the G protein–coupled receptor 3 (GPR3), a protein expressed in the brain, alleviated the cognitive deficits and reduced amyloid pathology in four different disease-relevant mouse models of AD. Furthermore, GPR3 was found to be elevated in postmortem brain tissue from a subset of AD patients. This study demonstrates that GPR3 is a potential AD therapeutic target and provides the validation needed for future development of GPR3 modulators. The orphan G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer’s disease (AD). However, given the vast resources required to develop and evaluate any new therapy for AD and the multiple failures involved in translational research, demonstration of the pathophysiological relevance of research findings in multiple disease-relevant models is necessary before initiating costly drug development programs. We evaluated the physiological consequences of loss of Gpr3 in four AD transgenic mouse models, including two that contain the humanized murine Aβ sequence and express similar amyloid precursor protein (APP) levels as wild-type mice, thereby reducing potential artificial phenotypes. Our findings reveal that genetic deletion of Gpr3 reduced amyloid pathology in all of the AD mouse models and alleviated cognitive deficits in APP/PS1 mice. Additional three-dimensional visualization and analysis of the amyloid plaque burden provided accurate information on the amyloid load, distribution, and volume in the structurally intact adult mouse brain. Analysis of 10 different regions in healthy human postmortem brain tissue indicated that GPR3 expression was stable during aging. However, two cohorts of human AD postmortem brain tissue samples showed a correlation between elevated GPR3 and AD progression. Collectively, these studies provide evidence that GPR3 mediates the amyloidogenic proteolysis of APP in four AD transgenic mouse models as well as the physiological processing of APP in wild-type mice, suggesting that GPR3 may be a potential therapeutic target for AD drug development.

Investigating Relaxation Processes in Cells and Developing Organisms: From Cell Ablation to Cytoskeleton Nanosurgery

Dynamic microscopy of living cells and organisms alone does not reveal the high level of complexity of cellular and subcellular organization. All observable processes rely on the activity of biochemical and biophysical processes and many occur at a physiological equilibrium. Experimentally, it is not trivial to apply a perturbation that targets a specific process without perturbing the overall equilibrium of a cell. Drugs and more recently RNAi certainly have general and undesired effects on cell physiology and metabolism. In particular, they affect the entire cell. Pulsed lasers allow to severe biological tissues with a precision in the range of hundreds of nanometers and to achieve ablation on the level of a single cell or a subcellular compartment. In this chapter, we present an efficient implementation of a picosecond UV-A pulsed laser-based nanosurgery system and review the different mechanisms of ablation that can be achieved at different levels of cellular organization. We discuss the performance of the ablation process in terms of the energy deposited onto the sample and compare our implementation to others recently employed for cellular and subcellular surgery. Above the energy threshold of ionization, we demonstrate how to achieve single-cell ablation through the induction of mechanical perturbation and cavitation in living organisms. Below this threshold, we induce cytoskeleton severing inside live cells. By combining nanosurgery with fast live-imaging fluorescence microscopy, we show how the apparent equilibrium of the cytoskeleton can be perturbed regionally inside a cell.