Geisel Lecture: Christopher Brett, Intraluminar Fragments

Samuel Reed ‘19

Intraluminar Fragments (ILFs) are bodies created within lysosomes during their fusion. This event has mainly been documented in yeast but is thought to be conserved among all eukaryotes, as is the case with other lysosomal pathways. ILFs were originally discovered by William Wickner, a Geisel professor, but little has been known of their function or related pathways. Christopher Brett, an Associate Professor at Concordia University in Montreal, recently visited Geisel and gave a lecture on his investigations into the topic.

The process of lysosomal fusion relies on tethering proteins and SNARE proteins, which bring lysosomes together. Tethering proteins create a connection between the organelles, while snare proteins are used later on to force the lysosomes to fuse. This process is regulated by Ypt7, a small GTPase and a homologue of Rab7. Like other GTPases, Rab7 activity is determined by the binding of the triphosphate-containing molectule GTP, and hydrolyzing the bond between the beta and gamma phosphates. During the fusion process, the membranes of the two organelles reach a state of “hemifusion” before the organelles are fully fused. In this hemifused state, the inner leaflets of the two plasma membranes come together to form a single membrane. Additionally, all the fusion proteins concentrate themselves at the point of hemifusion.

The hemifusion process is the key to ILF formation, or rather, to a lack thereof. In the models proposed by Brett, there are phases of ‘stalk’ formation, where the hemifused membrane spreads across the region of lysosomal contact, and ‘pore’ formation, where an opening in the membrane between the two organelles expands, breaking the hemifused region and causing the contents of the lysosomes to merge. Pore formation is carried out by a circle of fusion machinery, which entraps a region of plasma membrane that later becomes the ILF. However, if turnover between stalk formation and pore formation is too slow, there is no longer any non-hemifused membrane to be entrapped during pore formation, preventing an ILF from being formed. Although the exact model is not known, fusion machinery was thought to be involved in the turnover between the two processes.

These ideas were investigated using GTPγS, a non-hydrolyzable analog of GTP which constitutively activates Ypt7, and rVam7, a mutant SNARE protein that causes fusion regardless of Ypt7 activity. This was because, in a model the researchers had supported in the past, GDP-bound Ypt7 recruits tethering proteins, and GTP-bound Ypt7 recruits the SNARE proteins. As a result, GTPγS-bound Ypt7 results in lower fusion protein activity due to decreased Ypt7 tethering protein recruitment, while rVam7 does the opposite (1). Brett et al. found that with rVam7, ILFs were found with great frequency. With GTPγS, they were found much less frequently. They also found that lipid mixing – a measure of membrane fusion – and content mixing – a measure of pore fusion, occurred much closer together with rVam7, but that content mixing was delayed with GTPγS. This data collectively supports the idea that more tether and SNARE activity results in a faster turnover between stalk and pore formation, allowing the latter process to form an ILF. (1)

Brett discussed other experiments that supported this conclusion as well. In one particularly interesting example, cryo-ECM, an electron microscopy technique that uses samples fixed through freezing, was used to observe lysosomes at different stages of fusion. When lysosomes were observed with their membranes aligned with narrow, regular spacing, they were considered to be ‘docked.’ Docking is a state where the vesicles are touching after tethering protein activity, but where they haven’t yet fused. The measured distance between the docked lysosomes was 8 nm, the expected size of the tethering protein complex. This further emphasized the role of fusion machinery in ILF formation.

Research into ILFs is important for modern medicine. It is now well-documented that cancer cells have weakened lysosomes due to frequent autophagy, causing lysosomal overuse. This trait has been the target of many cancer drugs, which attack all of the body’s lysosomes, but only do real harm to the already-damaged cancer cell lysosomes. ILFs are thought to be part of a pathway for selectively recycling lysosomal proteins and are commonly seen in cancerous cells. More research into ILFs could be telling of lysosomal maintenance, and other knowledge of lysosomal pathways and functions.

References:

  1. Mattie, S., McNally, E. K., Karim, M. A., Vali, H., & Brett, C. L. (2017). How and why intralumenal membrane fragments form during vacuolar lysosome fusion. Molecular biology of the cell, 28(2), 309-321.

 

 

 

 

 

 

 

Source: Wikipedia (https://en.wikipedia.org/wiki/Lipid_bilayer_fusion)

Caption: Two organelles, such as lysosomes, fuse together with a hemifused intermediate. Early pore formation can interrupt hemifusion, resulting in an ILF.