Organite | EPFL Graph Search

Les organites (de temps en temps nommés organelles par anglicisme) sont les différentes structures spécialisées contenues dans le cytoplasme et délimitées du reste de la cellule par une membrane phospholipidique. Il existe de nombreux types d'organites, en particulier dans les cellules eucaryotes. On a longtemps pensé qu'il n'y avait pas d'organites chez les cellules procaryotes, mais quelques exceptions ont été mises en évidence. Principaux organites thumb|upright=1.5|Schéma d'une cellule animale type. Composants : (1) Nucléole
(2) Noyau
(3) Ribosomes
(4) Vésicule
(5) Réticulum endoplasmique rugueux (ou granuleux) (REG)
(6) Appareil de Golgi
(7) Cytosquelette
(8) Réticulum endoplasmique lisse
(9) Mitochondries
(10) Vacuole
(11) Cytosol (12) Lysosome
(13) Centrosome (constitué de deux centrioles) (14) Membrane plasmique

Le noyau (2), contient l'essentiel du matér

Fluorogenic probes for live-cell imaging

Aleksandar Salim

The development of novel fluorogenic probes and their use in live-cell imaging lead to a plethora of discoveries in biological research. Herein I report on novel red fluorogenic probes for live-cell super-resolution microscopy of various cellular organelles. First, I describe a new silicon rhodamine fluorophore termed SiR595. As most other SiR derivatives, SiR595 exists in an equilibrium between two chemical forms, a fluorescent zwitterion and a non-fluorescent spirolactone. Compared to regular SiR, the equilibrium of SiR595 is shifted towards the non-fluorescent, uncharged spirolactone. When coupled to appropriate targeting ligands, the resulting SiR595-based probes show high cell permeability. Furthermore, SiR595-based probes are fluorogenic as binding to their targets shifts the equilibrium towards the fluorescent zwitterion. I took advantage of these properties to develop novel red fluorogenic probes for live-cell imaging of Halo-tagged proteins, microtubules, F-actin and DNA. I was furthermore able to show that all of these probes are compatible with live-cell STED nanoscopy. Furthermore, I developed fluorogenic probes for live cell imaging of centrosomes. The centrosome is the principal microtubule-organizing center in animal cells. We targeted polo-like kinase 4 (Plk4), a serine/threonine-protein kinase, which localizes to centrioles throughout the cell cycle. To label Plk4 in live cells, we used Centrinone, a potent inhibitor of Plk4. Conjugating Centrinone to SiR595, I obtained and characterized several fluorogenic SiR595-Centrinone probes. SiR595-Centrinone probe labels overexpressed GFP-Plk4(K41M) during live-cell imaging and could be used to characterize the spatial distribution of GFP-Plk4(K41M) using live-cell STED nanoscopy. While the here introduced probes for Plk4 are not bright and fluorogenic enough to detect endogenous Plk4, the work provides the foundation for the development of such probes.

EPFL2020

High-throughput and adaptive super-resolution fluorescence microscopy of organelles

Dora Mahecic

Fluorescence super-resolution microscopy has allowed unprecedented insight into the workings of biological systems below the diffraction limit of light. Over the past decade, it has overcome several challenges to deliver 3D, multi-color and faster imaging techniques, which have found many applications in deciphering the workings and structure of organelles â specialized cellular compartments whose shape and dynamics serve their specific functions. However, most super-resolution techniques are laborious and slow, and there are existing challenges in achieving sufficient throughput for collection of large datasets and capturing dynamic processes in living cells. The aim of this thesis is to address issues of limited throughput, temporal resolution and live-cell compatibility, with application to centriole architecture and mitochondrial dynamics. The design of a novel flat-fielding module for multi-focal excitation which â once integrated into an instant structured illumination microscope â allows the capture of larger fields of view with uniform illumination and hence results in increased throughput. This flat-fielded microscope allows multi-color, 3-dimensional imaging at double the diffraction-limited resolution of up to 100 mammalian cells within 1-2 minutes. Furthermore, combined with advanced sample preparation using expansion microscopy, the setup achieves an effective resolution of ~35 nm, comparable to some of the more powerful super-resolution techniques, but at 100-1000 times increased throughput. To showcase this improvement, we study the distribution of post-translational modifications within the centriole, revealing previously unobserved molecular organization. Second, with fast, live-cell imaging, we investigate the role of membrane bending energy and tension in mitochondrial division. While many molecular factors involved in this process have been identified, little is known about its physical requirements. By analyzing mitochondrial constrictions enriched in Drp1 that divide successfully or reverse, we establish that mitochondrial constrictions with higher bending energy are more likely to divide. Furthermore, by analyzing changes in mitochondrial shape and employing a novel membrane tension sensor, we observe that conditions under which mitochondrial membrane tension is increased, result in more successful divisions. These observations are supported by a model in which membrane bending energy increases the elastic energy at the constriction site, bringing it closer to the energy barrier to fission, while membrane tension acts as a fluctuation to overcome the remaining residual energy. Finally, to overcome existing trade-offs between temporal resolution and imaging duration, we develop an acquisition framework for smart and adaptive temporal sampling. By adapting the temporal resolution is in response to events of interest, they are captured at high imaging speed, while preserving the sample during a lack thereof. Using the accumulation of Drp1 to detect mitochondrial constriction sites allowed us to adapt the imaging speed to capture mitochondrial constrictions at high temporal resolution while preserving the sample when such events are lacking. Overall, this framework provides both high temporal resolution and long imaging times when needed, decreasing resulting phototoxicity, photobleaching and amount of redundant data.

EPFL2020

Fluorogenic probes for live-cell imaging

Aleksandar Salim

EPFL2020

High-throughput and adaptive super-resolution fluorescence microscopy of organelles

Dora Mahecic

EPFL2020