The Botelho Lab @ Ryerson University in Toronto studies the molecular mechanisms that establish, maintain or adapt organelle identity and function. Organelles underpin everything that cells perform. Thus, understanding how organelles are made and function is essential to comprehend the underpinnings that drive life, health and disease- states. You are invited to explore and learn about our work and our energetic group. Please contact Dr. Roberto Botelho if interested in graduate or post-doctoral studies.
Lysosomes in dendritic cells exposed to monophosphoryl lipid A
Immune cells like macrophages and dendritic cells posses lysosomes that look like little globules under the fluorescence microscope. However, once these cells are activated by exposure to bacterial products or other immune triggers, these lysosomes change! For one thing, we showed that phagocytosis enhances expression of lysosomal proteins to bolster lysosomal activity, making macrophages better killers (Gray et al. ). In other words, phagocytosis trains macrophages to be better killers. For another, lysosomes are transformed from many vesicular structures to a highly tubular network (see Saric et al.). This tubulation process may play a role in retaining and processing antigens, which the immune system then uses to “engineer” antibodies (literally) against that antigen. We have recently published a review in Cellular Microbiology (Hipolito et al) that summarizes lysosome remodeling and adaptation in immunity.
RAW 2FYVE GFP phagocytosing legionella filament
Macrophage eating filamentous Legionella (red). The macrophage is expressing 2FYVE-GFP, a fluorescent protein probe that tracks the dynamics of phosphatidylinositol-3- phosphate, a lipid that controls membrane trafficking. (In collaboration with Dr. Mauricio Terebiznik, U. of Toronto)
Macrophages, neutrophils and dendritic cells are immune cells that hunt down and engulf pathogens using their plasma membrane in a process called phagocytosis. After engulfment, the pathogen is sequestered in a new membrane-bound organelle called the phagosome. Importantly, the phagosome is then converted into a phagolysosome, a process known as phagosome maturation. This transformation changes the phagosome from an innocuous organelle to a very acidic and hydrolytic organelle that kills and digests the pathogen (see Gray & Botelho for recent review. Studying this system has two great advantages. First, this is a critically important process in the immune arsenal against pathogens – and one that can be hijacked by bugs like Salmonella and Mycobacterium. Second, the phagosome is a beautiful system to study organelle identity processes because we can track an organelle from birth (phagocytosis) to death (phagosome resolution) and study the changes in its identity over many hours. Currently, we are exploring how phosphoinositides regulate phagosome maturation (eg. Dayam et al., 2017), how cells adapt to phagocytosis (example: Gray et al. ), and how macrophage handles challenging particles like filamentous Legionella (eg. Naufer, Hipolito et al - a great collaboration with the Terebiznik lab at U of Toronto). Not yet published, we are also investigating what the heck happens to phagosomes after bacteria are digested!
Cells treated with PIKfyve inhibitors display massively swollen lysosomes (red). Green is a new GFP-probe for phosphatidylinositol-3- phosphate.
We are spending buckets of time trying to better understand the regulation and function of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], a phosphoinositide that controls properties of lysosomes and the yeast vacuole. As mentioned earlier, lysosomes are responsible for degrading macromolecules, organelles, and pathogens and recycle the raw materials to use as nutrients. They also can serve as storage organelles. When cells cannot synthesize PtdIns(3,5)P2, lysosomes become massively swollen, autophagic flux is impaired, phagosome maturation is arrested, even neutrophil chemotaxis is blunted, organisms fail to develop properly, there is a burst of inflammation, and neurological problems arise. So arguably, PtdIns(3,5)P 2 kind of matters. For recent work on this lipid by our lab see Ho et al . We are now trying to understand why lysosomes become massively enlarged and how this lipid coordinates with transcription factors that modulate lysosome biogenesis.
Finally, we typically recognize that there are seven phosphoinositide (PIP) lipids based on the phosphorylation state of the headgroup (like PtdIns(3,5)P2 above). However, PIPs are phospholipids and thus contain two fatty acid tails. Interestingly, PIPs are enriched for two types of fatty acids: stearate, which is 18 carbons long and saturated, and arachidonic acid, an omega-6 lipid that is 20 carbons long and unsaturated. In collaboration with the Antonescu lab , we are now dabbling on how acylation specificity matters (see Bone et al., 2017; ). Yet, there stearate and arachidonic acid are not the sole chains that are used in PIPs. This differential acylation could generate functionally distinct pools for each phosphoinositide (see Choy et al for recent review on PIP biology). We are also dabbling on this.
Downtown Toronto, also known as centre of the Universe (:p). That means you can travel the world within a few blocks, including eat burritos for breakfast, sashimi for lunch and vindaloo for dinner, go shopping, see a movie or a ballet, visit a museum, go skating, go for a walk to o chill by the lake or The Toronto Islands, take a boat cruise, climb the CN Tower, go see a game (even the Leafs), and feed pigeons and squirrels (if that's your thing).: The Botelho Lab is found in
and the Department of Chemistry and Biology are the academic and administrative home for the Botelho Lab. For quick facts on Ryerson and how much it has grown see here. Graduate students in the lab are enrolled in Molecular Science Graduate Program . I am always looking for committed and driven students
Ryerson MaRS Facilities. These state-of-the art research and study facilities were inaugurated in August 2016 and will bring together about 10 research labs in the Department of Chemistry and Biology. We got the goodies that you need to do your work, places to study and chill, and the views (11th floor) of Queen's Park, downtown and that other University (and the avenue).The Botelho Lab is housed in
From Halloween door competition to ski trips to ale-time and holidays parties. We try to do things together (though admittedly this requires a party planner!). Below shows some activities we have done over the years... Antonescu group is often a partner in crime.