Botelho Lab

mmune cells like macrophages and dendritic cells possess 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 (Gray et al.) . In other words, phagocytosis trains macrophages to become better killers. For another, lysosomes are transformed from many vesicular structures to a highly tubular network (see Saric et al. , for example). This transformation is also accompanied by expansion of the lysosome size through enhanced translational activity driven by mTORC1 (see Hipolito et al. ). Lysosome tubulation and expansion seems to play a role in antigen presentation, which the immune system then uses to “engineer” an attack plan on the source of antigens. For more information on this processes, see recent reviews from our lab in Cellular Microbiology (Hipolito et al LINK PMID: 29349904) and in Frontiers (Inpanathan and Botelho, LINK PMID: 31281815). Currently, we are how else lysosomes adapt during phagocyte activation, how lysosome remodelling matters for the immune response, and how the translational regulatory machinery aids to reorganize the endomembrane system.

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!

We are investing resources 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)P2 kind of matters. For recent work on this lipid by our lab see Ho et al and Choy, Saffi et al. We are now trying to understand how lysosomes become massively enlarged and how this lipid coordinates a variety of seemingly unrelated processes to lysosomes including gene expression mechanisms.

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 (link), we have been working to understand how cells establish this acyl profile and how this impacts cellular functions (see Bone et al., 2017 ). Current research projects are now focused on understanding i) how PIP acyl profile is important for cell signalling, cancer proliferation, and metastasis and ii) the mechanisms that establish PIP acyl profile. For more detailed discussion on these topics, please see Choy et al for a recent review on PIP biology.