The Langlais Lab is all about insulin signaling, more specifically, discovering the underlying mechanisms governing insulin-stimulated glucose uptake. Through a series of protein signal transduction pathways, insulin stimulates the translocation of the insulin-sensitive glucose transporter GLUT4 to the cell surface, resulting in a massive influx of glucose into target cells in both muscle and adipose tissue. The net result is the lowering of blood glucose levels back to normal, a process that if interfered with (termed “insulin resistance), can be a contributing factor to the pathogenesis of type 2 diabetes. So we attack this problem with the mantra that you can’t fix it until you know how it works.
In 2011, by taking advantage of affinity purification mass spectrometry (AP-MS), Dr. Langlais, as a post-doctoral fellow in Dr. Mandarino’s lab, discovered that insulin stimulates the phosphorylation of the microtubule associated protein, CLASP2, a finding that eventually tied CLASP2 to insulin-regulated trafficking of GLUT4 and glucose uptake. Since CLASP2 tracks along the growing plus-end of the microtubule, a hypothesis evolved that proposes CLASP2 directs growing microtubules to hotspot landing zones on the plasma membrane, whereby setting up accessible GLUT4 for acute translocation and plasma membrane fusion (Langlais et al, JBC, 2012). The Langlais Lab has been in hot pursuit of this notion ever since.
The first big move on the CLASP2 project started as “just characterizing the CLASP2 interactome”, which turned into a 5+ year gauntlet. Turns out, pulling off an actual conclusive interactome is difficult, but even worse, is trying to avoid false positives. Once those hurdles were overcome, a chain of reciprocal interactome experiments led to the establishment of a CLASP2 protein network in adipocytes, and also niftily managed to identify a new microtubule-associated protein, SOGA1 (Kruse et al, MCP, 2017).
Next up we set out to test the hypothesis that insulin affects CLASP2 dynamics in a live-cell setting. For this, we collaborated with the Mouneimne Lab and Sara Parker, a Post-Doctoral Fellow, who ended up joining in on the obsession. We were able to link CLASP2 together with G2L1, a plus-end binding protein that associates with both microtubules and actin, and we discovered that insulin causes both proteins to spatially reorient themselves along microtubules. The microtubules looked braced with all that CLASP2 and G2L1 after insulin stimulation, an observation that once we followed up on, led to the discovery that insulin causes microtubules to stabilize, a finding we brought Nam Lee's Lab in to help out with.
The cherry on top of this scientific smorgasbord though was another hypothesis we had that hit pay dirt. We did not think CLASP2 was the only microtubule associated protein that responds to insulin stimulation, so using a novel label-free mass spectrometry-based method for quantifying directional changes in protein phosphorylation that we came up with, we discovered that the CLASP2 protein network members MARK2, CLIP2, G2L1, AGAP3, CKAP5, and the closely associated EB1, all undergo insulin-regulated phosphorylation. We now believe that it is not just CLASP2, but rather, a network of microtubule-associated proteins that synergize to coordinate insulin-regulated microtubule dynamics. All this ended up being wrapped up together in our latest publication (Parker at al, 2019, MCP).
What’s next right? We are currently wrapping up our project on characterizing insulin-regulated CLASP2 phosphorylation. We are also jumping in to the world of live-cell GLUT4 trafficking to test if these microtubule-associated proteins and GLUT4 are spatially related and functionally connected.