CryoEM of frozen membranes has revealed its most intimate folds. Biochemistry of tissue extracts says it bundles up with other secretases. But how does γ-secretase, the all-important enzyme that cleaves Aβ42 from its precursor, truly behave in the cell membrane? In the July 9 eLife, researchers led by Wim Annaert at KU Leuven, Belgium, offer a nanoscopic glimpse of this wily intramembrane protease. Adopting various models of super-resolution light microscopy, they spied the secretase gliding through the plasma membrane alone or in pairs, but almost never in larger complexes. It lingered with substrates, but rarely dallied with other secretases. The protein complex occasionally stopped by ‘hotspots,” possibly characterized by a distinct lipid microenvironment, where it may be less active.
- Super-resolution microscopy tracks individual γ-secretase complexes.
- Protease travels the cell membrane alone or in pairs.
- No evidence for megadalton super complexes with other secretases.
“Super-resolution microscopy is the type of technology we need to move the field forward and better understand how and where intramembrane proteolysis occurs,” Annaert told Alzforum. “This study is cutting-edge, and the authors have performed a robust analysis of the data,” wrote Gopal Thinakaran, University of South Florida, Tampa.
First author Abril Angélica Escamilla-Ayala and colleagues used a slew of rainbow-colored, fluorescently tagged proteins to simultaneously track γ-secretase subunits and substrates at single-molecule resolution in the cell membranes of mouse embryonic fibroblasts. This allowed them to visualize, in real time, the movement of these proteins in a way that has never been achieved before.
“This is an important technical first step toward investigating the lateral surface diffusion of γ-secretase particles in neuronal synapses, to better understand the structural and functional dynamics of this protease in the context of synaptic activity and synaptic plasticity, both in healthy and disease/AD states,” wrote Patrick Fraering, Foundation Eclosion, Plan-les-Ouates, Switzerland.
First, the authors established the stoichiometry of the complex’s subunits using fluorescent presenilin (PS)1 and nicastrin (NCT) chimeras. They introduced these into fibroblasts that had their own PS1, PS2, and nicastrin genes deleted. Escamilla-Ayala checked that that the fluorescent PS1 and NCT formed functional γ-secretase complexes with endogenous PEN2 and Aph1, the other two components of the γ-secretase tetramer. Then she used Structured Illumination Microscopy (SIM) to determine the PS1-NCT ratio of γ-secretase complexes on the cell surface. The scientists examined plasma membrane sheets supported on a silica matrix to eliminate interference from complexes in the Golgi, endosomes, or other subcellular organelles. They found that most PS1 molecules in the plasma membranes were within 50 nM of one NCT molecule, and vice versa. This indicates that each subunit binds in a 1:1 ratio, since a second partner would lie further away. The finding confirms, in this type of living cell, the 1:1 ratio found in prior biochemical analyses. It is in keeping with the accepted stoichiometry of one each of the four γ-secretase subunits per mature protease.
Next, the authors looked in the plasma membrane sheet for higher-order organization. Previously, researchers working in Dennis Selkoe’s lab at Brigham and Women’s Hospital, Boston, reported that they had isolated megadalton-sized complexes comprising multiple molecules of APP, α-, β, and γ-secretase from mouse brain extracts (Jan 2019 news). Could these mega-complexes be seen at the plasma membrane?
Complex Squiggles. Analyzing tracks (colored lines, left) made by individual γ-secretase complexes as they travel through the cell membrane reveals “hotspots” (white ovals, right), where complexes meet. [Courtesy of Escamilla-Ayala et al., eLife 2020.]
Escamilla-Ayala and colleagues found no evidence for this. A next-nearest neighbor analysis of SIM data indicated that nicastrin molecules tagged with two different fluorophores were randomly distributed and rarely came into contact with each other, suggesting γ-secretase travels by itself (see image above). Other evidence supported this. For example, it took only one photobleaching step to quench fluorescence of PS1 chimeras in the complex. This means that each contained but one fluorophore—hence only one PS1. In living cells, the authors found no evidence for mega complexes, either. Here they used a super-resolution technique called PhotoGate to bleach an area, then watched while fluorescent molecules repopulated it. By measuring the intensity of the fluorescence of the molecules diffusing into the field of view, the researchers were able determine how many presenilin molecules they contained, and hence how many γ-secretase complexes. They found that about 60 percent of γ-secretase diffused as a monomer and 40 percent as a dimer; there were hardly any higher-order structures.
What about complexes with substrates? Or with other secretases? Using fluorescent antibodies and fluorescent chimeras, the authors checked to see how close γ-secretase cozied up to two of its substrates, APP and N-cadherin, and to the secretases ADAM10 and BACE1. Next-nearest neighbor analysis of SIM data showed that γ-secretase associated with its substrates, but not with ADAM10 or BACE1.
Likewise, a method called single-particle tracking coupled to photo-activated localization microscopy (SptPALM) offered little indication that secretases formed mega complexes. SptPALM tracks individual molecules as they diffuse through the cell membrane and can superimpose those tracks to determine when molecules interact (see image above). Escamilla-Ayala saw that γ-secretase and BACE1 often crossed paths. Alas, when they did their diffusion properties stayed the same, suggesting that they were merely passing each other like so many ships in the night. On the other hand, γ-secretase seemed to linger as it passed ADAM10 hotspots, but while this could in theory indicate that they interact, the authors think it’s more likely that both merely stop at the same “hotspot” in the membrane on occasion, possibly to “transfer” a substrate for the final proteolysis step, Annaert thinks (see image below).
Loner Complex. Hotspots for presenilin (green) do not overlap with those for ADAM10 (purple, left) or BACE1 (purple, right). [Courtesy of Escamilla-Ayala et al., eLife 2020.]
What those hotspots are remains a mystery. The authors found that almost 90 percent of γ-secretase complexes are mobile in the membrane, albeit not all are traveling at the same speed. This heterogeneity might reflect different conformational states or interactions with various membrane components, they believe. Using inhibitors to lock γ-secretase in a “compact” conformation it adopts while cleaving substrates did not affect diffusion, suggesting that active secretase moves about the membrane just as much as inactive does.
Despite the motility, different secretase complexes tended to visit the same hotspots, where they often paused. Annaert is trying to characterize these spots. They may contain markers of lipid rafts, implying they might provide a specific environment. Interestingly, because γ-secretase inhibitors (GSIs) reduced the number of hotspots, the authors believe they are not “hot” in terms of γ-secretase activity; instead, they may be places where the inactive complex briefly rests.
How to reconcile this new data with reports of secretase megacomplexes? “We believe a major difference in the Escamilla-Ayala et al. study is their use of exogenous expression of tagged proteins instead of genome-edited endogenous tagging,” wrote Selkoe and Lei Liu, also from Brigham and Women’s, in a comment to Alzforum (see below). “Exogenous expression of Presenilin-1, Nicastrin, BACE1, and ADAM10 could lead to unavoidable artifacts,” they added. Annaert told Alzforum that his group went to great pains to rule out overexpression artifacts. Fraering agreed. “It should be noted that, in this study, the authors paid special technical attention to minimize the potential risk of overexpression artifacts,” he wrote.
Whether the same findings reported here will hold true in neurons remains to be seen. This study is a first step toward using these methods in more relevant cell types. “Now that we have the right constructs and know the best fluorescent tags, we plan to study γ-secretase in synapses, in dendrites, and in neurites to see what the effects are of APP mutations, inhibitors, and other molecules—not only on activity but also on localization and diffusion of the protease,” said Annaert. “Then we can really understand how genetics affects cell biology in Alzheimer’s disease.”—Tom Fagan
- Escamilla-Ayala AA, Sannerud R, Mondin M, Poersch K, Vermeire W, Paparelli L, Berlage C, Koenig M, Chavez-Gutierrez L, Ulbrich MH, Munck S, Mizuno H, Annaert W. Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface. Elife. 2020 Jul 7;9 PubMed.