Olof Idevall-Hagren

Ongoing projects:

Organelle communication via membrane contacts sites (MCS)

Extended-Synaptotagmins generate ER-PM contacts

Optogenetic tool development and implementation

Plasma membrane PI(4,5)P2 regulate insulin secretion

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Organelle communication via membrane contacts sites (MCS)

The architecture of the prototypic mammalian cell has been the focus of intense study since the early days of microscopy. With the development of electron microscopy for the biological sciences in the 1950’s came the detailed characterization of most cellular organelles, like the endoplasmic reticulum (ER), the Golgi apparatus and secretory vesicles. More recently, using live cell imaging techniques, it has been found that these organelles are highly dynamic structures that constantly reform, reshape and redistribute within the cell. Many organelles also seem to communicate through direct contacts, formed by protein and lipid complexes. Such membrane contact sites (MCS) are hubs for the transfer of lipids, ions and proteins between the organelles and key to the normal function of these cellular compartments. With the insulin secreting pancreatic ß-cell as our model, we employ high-resolution fluorescence microscopy together with genetically encoded biosensors and molecular tools to study and manipulate these cellular structures in order to better understand their function.

Figure 1: Schematic drawing depicting an insulin-secreting pancreatic b-cell. The membrane-enclosed endoplasmic reticulum (ER) of these cells is distributed throughout the cytosol of the cell and makes contacts with numerous other membranes, including the plasma membrane (PM) and the mitochondria (Mito.). Numerous processes occur at these membrane contact sites (MCS) but little is known about their composition.


Extended-Synaptotagmins generate ER-PM contacts

We have characterized a family of proteins called the Extended-Synaptotagmins (E-Syts). These ER-anchored proteins bind to the plasma membrane upon changes in the cytosolic Ca2+ concentration (see Figure 2), thus generating physical connections between the two membranes. We are currently trying to understand the biological function of these proteins.

Figure 2: A. Electron micrograph of a fibroblast where the ER is stained black. Notice how parts of the ER are in close proximity of the cell periphery (red box). B. Schematic illustration depicting Extended-Synaptotagmin-1 (E-Syt1), an ER-anchored protein that also binds the plasma membrane by interactions with specific lipids. C. Confocal microscopy images of a very flat cell expressing fluorescence-tagged E-Syt1 under conditions where the cytoplasmic Ca2+ concentration is low or high. Notice how E-Syt1 aggregate at the plasma membrane when the Ca2+ concentration increases.


Optogenetic tool development and implementation

Optogenetics is the modification and use of light-regulated proteins, typically isolated from plants or bacteria, to enable control of cellular processes by illumination. Expression of optogenetic tools has for example enabled light-dependent control of neurotransmitter release, insulin release, cell migration and transcription. We have previously used light-dependent protein-protein interactions to recruit lipid synthesizing and degrading enzymes to the plasma membrane, leading to the discovery that rapid changes in lipid levels can polarize cells and is sufficient to induce e.g. directed cell migration. Current work aims at using these optogenetic tools to generate inducible contacts between various cellular organelles and determine how this affects cell function. Since optogenetics is a non-invasive technique, we also work on adapting it to in vivo settings.

Figure 3: A. Drawing showing the principle of light-induced heterodimerization. One part of the optogenetic module (CIBN) can be anchored to any cellular membrane (target membrane) whereas the other part (CRY2) can be fused to a protein of interest (here a Red Fluorescent Protein). Blue-light illumination promotes the interaction between CIBN and CRY2 and causes redistribution of the protein of interest to the target membrane. B. Focal blue-light illumination (blue square) allows recruitment of a lipid-degrading enzyme (green) to a restricted part of the plasma membrane, resulting in corresponding loss of a specific lipid (red).


Plasma membrane PI(4,5)P2 regulate insulin secretion

PI(4,5)P2 is a rare lipid in the plasma membrane known as a master regulator with targets including components of receptor signaling, endo- and exocytosis and ion channels. In recent work, using optogenetic depletion and synthesis of PI(4,5)P2, we found this lipid to control voltage-dependent Ca2+ influx in ß-cells. Light-induced removal of PI(4,5)P2 acutely blocked Ca2+ increases induced by glucose, resulting in strong inhibition of insulin secretion (see Figure 4). We are currently investigating additional roles of this lipid in the regulation of ß-cell function.

Figure 4: Traces showing measurements of the cytosolic Ca2+ concentration in glucose-stimulated b-cells. In control cells (left), blue-light illumination did not alter the regular Ca2+ oscillations, whereas these were completely abolished in cells expressing a light-regulated PI(4,5)P2 phosphatase (right).


Methods

Total internal reflection fluorescence microscopy, confocal microscopy, optogenetics, image analysis, cell culture, molecular biology, ELISA, Western blot.


More info on established methods,  instruments and techniques  available in the group.


For further information about this research group please contact
Olof Idevall-Hagren