Alla Kress

Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

Mapping the Local Organization of Cell Membranes Using Excitation-Polarization-Resolved Confocal Fluorescence Microscopy

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.

Probing Orientational Behavior of MHC Class I Protein and Lipid Probes in Cell Membranes by Fluorescence Polarization-Resolved Imaging

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane. We apply this methodology on cells after treatments affecting the actin and microtubule networks. We find in particular opposite orientational order changes of proteins and lipid probes in the plasma membrane as a response to the cytoskeleton disruption. This suggests that MHC I orientational order is governed by its interaction with the cytoskeleton, whereas the plasma membrane lipid order is governed by the local cell membrane morphology.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Significance Central biological processes in cells and tissues are intrinsically governed by the structural order of biomolecular assemblies. It is thus a key factor to decipher how these assemblies organize in complex molecular organizations, from the nanometric to the macroscopic scale. Polarized microscopy can access such information; however, signals are spatially averaged over the optical diffraction limit and are contaminated by the fluorophores’ orientational flexibility of their linker to the biomolecules. By bringing polarized fluorescence down to superresolution microscopy using single-molecule localization, we show that structural imaging can be scaled down to nanometric scales and is able to discriminate fluorophores’ flexibility from biomolecules’ orientational order. We demonstrate nanoscale structural imaging in fundamental biological filament organizations. Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.

Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores.

The orientational distribution of fluorophores is an important reporter of the structure and function of their molecular environment. Although this distribution affects the fluorescence signal under polarized-light excitation, its retrieval is limited to a small number of parameters. Because of this limitation, the need for a geometrical model (cone, Gaussian, etc.) to effect such retrieval is often invoked. In this work, using a symmetry decomposition of the distribution function of the fluorescent molecules, we show that polarized two-photon fluorescence based on tunable linear dichroism allows for the retrieval of this distribution with reasonable fidelity and without invoking either an a priori knowledge of the system to be investigated or a geometrical model. We establish the optimal level of detail to which any distribution can be retrieved using this technique. As applied to artificial lipid vesicles and cell membranes, the ability of this method to identify and quantify specific structural properties that complement the more traditional molecular-order information is demonstrated. In particular, we analyze situations that give access to the sharpness of the angular constraint, and to the evidence of an isotropic population of fluorophores within the focal volume encompassing the membrane. Moreover, this technique has the potential to address complex situations such as the distribution of a tethered membrane protein label in an ordered environment.

A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell.

A fluorescence correlation spectroscopy system based on two independent measurement volumes is presented. The optical setup and data acquisition hardware are detailed, as well as a complete protocol to control the location, size, and shape of the measurement volumes. A method that allows to monitor independently the excitation and collection efficiency distribution is proposed. Finally, a few examples of measurements that exploit the two spots in static and/or scanning schemes are reported.

Imaging Molecular Order in Cell Membranes by Polarization-Resolved Fluorescence Microscopy

The use of light polarization properties in the analysis of fluorescence images has driven a large amount of research toward the measurement of orientational behavior of molecules in cells, in particular in their membranes. This field has been recently revisited to enlarge the possibilities of polarization-resolved fluorescence microscopy. We show that this technique allows retrieving a wealth of information on the constraints that hinder rotational mobility of lipid probes and proteins in membranes, bringing thus new insights on inter-proteins and lipid-protein interactions, on membrane morphology at the sub-diffraction length scale and on local membrane physical properties such as viscosity.

High frame-rate fluorescence confocal angle-resolved linear dichroism microscopy

Angle-resolved linear dichroism is a recent technique that exploits images recorded using an illumination field whose polarization angle is sequentially rotated during acquisition. It allows to retrieve orientation information of the fluorescent molecules, namely the average orientation angle and the amplitude of the fluctuations around this average. In order to boost up the acquisition speed without sacrificing the axial sectioning, we propose to combine a spinning disk confocal excitation scheme together with an electrooptical polarization switching and a camera acquisition. The polarization distortions induced when passing through the spinning disk system have been quantified and effectively compensated. The signal to noise features of the camera have been analyzed in detail so that the precision of the method can be quantified. The technique has been successfully tested on giant unilamellar vesicles and on living cells labeled with different fluorescent lipid probes, DiIC18 and di-8-ANEPPQ. It was able to acquire precise orientation images at full frame rates in the range of a second, ultimately limited by the fluorophore brightness and the camera sensitivity.

Lipid Order Investigations Combined with Generalized Polarization Provide Deeper Insights into Plasma Membrane Architecture of Live Cells

The complexity of cell plasma membrane architecture is governed by biomolecular interactions, heterogeneity and complex sub-micrometric size domains. Even though there is no doubt about the existence of liquid-ordered and liquid-disordered phases in the model membranes, direct observations in live cells remain delicate due to their dynamic nature and sub-resolution size.High resolution optical microscopy techniques have helped the understanding of lipid organization and their contribution to biological functions. Among them, environmentally sensitive lipid probes (such as Laurdan or derivatives of ANEP) reveal lipid packing information (in particular fluidity governed by local polarity) at the nanometer scale, thanks to the bathochromic shift in their fluorescence emission spectrum as the membrane undergoes phase transition from gel to fluid [1]. The associated spectral ratiometric imaging is called generalized polarization (GP).In this work, we combine GP imaging with orientational investigations on such lipid probes, in order to probe, in a complementary approach, both local lipid packing (which reports a molecular scale information) and lipid order (which report a mesoscopic scale information). The orientational order of lipid probes, measured by fluorescence angle-resolved linear dichroism microscopy (FARLDM), has been previously shown to be highly sensitive to local membrane morphological changes (driven by cytoskeleton alterations) and cholesterol depletion [2].Implementing a combined GP-FARLDM technique, we show that new information can be gained on lipids interactions at different scales in the cell plasma membrane. We show in particular that liquid-ordered and liquid-disordered phases exhibit distinct morphological behaviors, and explore the capacities of different lipid probes to report information on the membrane architecture at the nanoscale.References[1] Gaus et al. (2003) Proc. Nat. Acad. Sc. 100:15554-15559.[2] Kress et al. (2013) Biophys J 105:127-136.

Imaging molecular organization of cell membranes and proteins assemblies using polarimetric fluorescence microscopy

Biomolecular orientational organization is an important parameter in biological processes where functions (such as cell motility, vesicular trafficking, signalling, protein interactions, etc.) are closely related to orientation and ordering. The investigation of structural behaviors is today a determining factor for a better understanding of fundamental mechanisms governing cell membranes and proteins assemblies, which is particularly relevant for instance in neuro-degenerative diseases related to proteins aggregates formation.