Dataset Information

Mass spectrometry-based proteomic analysis of the liprin-α1 interactome in MIN6 cells

ABSTRACT: Accumulating evidence suggests that insulin secretion in pancreatic β cells is regulated by mechanisms analogous to synaptic vesicle release at the neuronal presynaptic active zone (AZ). Neurotransmitter release is tightly regulated in magnitude, space and time by a presynaptic macromolecular complex including scaffold proteins liprin, ELKS, RIM and RIM-BP, to ensure precise control of synaptic responses. This presynaptic complex tethers, docks and locally controls the fusion of synaptic vesicles at the AZ. β-cells express many of the same neuronal presynaptic scaffold proteins including liprin-α1, however, no examination into the existence of a β cell presynaptic-like complex has been done. Here, we performed the first comprehensive interactome analysis of liprin-α1 in MIN6 cells using co-immunoprecipitation coupled to mass spectrometry-based proteome analysis to identify liprin-α1 binding partners and probe the existence of a wider β cell presynaptic-like complex. We identified a broad range of liprin-α1 interacting proteins in both basal (low glucose) and stimulated (high glucose) conditions. Key among these were the synaptic proteins liprin-α1,2, liprin-β1, GIT1 and LAR, supporting a β cell presynaptic-like complex. Other proteins of interest included β2 syntrophin, a regulator of insulin granule mobility, the microtubule organising proteins Mtcl1 and Mtcl3, as well as protein phosphatase 2A complexes and the functionally related 14-3-3 isoforms involved in phospho-protein dependent binding. Together, these data indicate liprin-α1 is central to a wider presynaptic-like complex that is the focus for phosphorylation and structurally links insulin granule positioning and cytoskeletal dynamics.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Mark Larance

LAB HEAD: Mark Larance

PROVIDER: PXD049219 | Pride | 2026-02-09

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20230921_Exploris480_ML_KD_EXP2_TUBE1_GFP_BASAL.raw	Raw
	20230921_Exploris480_ML_KD_EXP2_TUBE2_LIPRIN_BASAL.raw	Raw
	20230921_Exploris480_ML_KD_EXP2_TUBE3_GFP_STIM.raw	Raw
	20230921_Exploris480_ML_KD_EXP2_TUBE4_LIPRIN_STIM.raw	Raw
	20231129_Exploris480_ML_KD_EXP3_GFP_BASAL.raw	Raw

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Publications

Submembrane liprin-α1 clusters spatially localize insulin granule fusion.

Deng Kylie K Sun Kitty K Hallahan Nicole N Gan Wan Jun WJ Cielesh Michelle M Mahyad Baharak B Kebede Melkam A MA Larance Mark M Thorn Peter P

The Journal of cell biology 20250828 10

Insulin granule fusion in pancreatic β cells localizes to where they contact the ECM of the islet capillaries. The mechanism(s) underpinning localization are unclear. Using glucose or high K+ stimulation or the global uncaging of Ca2+, we show granule fusion consistently focused to the β cell-ECM interface, suggesting a specific localization mechanism. We tested for the involvement of liprin-α1, a scaffold protein enriched at the β cell-ECM interface. Liprin-α1 knockdown did not affect high K+-s ...[more]

PMID: 40875978

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Project description:Liprin-α1 is a widely expressed scaffolding protein that regulates cellular processes such as cell motility and synaptic transmission through assembly of localized higher-order molecular complexes. The dynamic regulation of these complexes remains poorly understood. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins into cellular nanodomains, facilitating efficient spatiotemporal signaling. Whether liprin-α1 undergoes regulated LLPS remains unclear. MS-based interactomics identified PPP2R5D, the regulatory B56δ subunit of PP2A, as a liprin-α1 interaction partner through a canonical short linear motif (SLiM) in its N-terminal dimerization domain. Mutation of SLiM4 nearly abolished liprin-α1 interaction with PP2A holoenzyme and resulted in a significant increase in GFP-liprin-α1 LLPS in HEK293 cells. Consistently, GFP-liprin-α1 exhibited increased droplet formation in PPP2R5D knockout HEK293 cells. Phospho-analysis of liprin-α1 SLiM4 mutant via MS revealed increased phosphorylation of multiple Ser/Thr sites, including S763, as validated by a novel phospho-specific antibody. A liprin-α1 S763E phospho-mimetic mutant appeared sufficient to drive LLPS. Expression of the PPP2R5D missense variant E420K, recurrently found in Houge-Janssens Syndrome Type 1 compromised suppression of liprin-α1 LLPS, correlating with increased liprin-α1 S763 phosphorylation. Mechanistically, a liprin-α1 E942A mutant unable to bind liprin-β1 underwent increased LLPS, despite preserved PPP2R5D holoenzyme binding. Furthermore, liprin-α1/β1 heterodimerization significantly decreased under conditions where liprin-α1 LLPS was promoted, i.e. upon SLiM4 or S763E mutation in wild type cells, or in PPP2R5D knockout and PPP2R5D E420K knock-in cells. Our findings identify both liprin-β1 and PPP2R5D-PP2A as potent inhibitors of liprin-α1 LLPS, with PP2A contributing to liprin-α1/β1 heterodimerization via phosphorylation of at least liprin-α1 S763.

Project description:This is a model of one presynaptic and one postsynaptic cell, as described in the article: Gamma oscillation by synaptic inhibition in a hippocampal interneuronal network model. Wang XJ, Buzsáki G. J Neurosci. 1996 Oct 15;16(20):6402-13. PMID:8815919; Abstract: Fast neuronal oscillations (gamma, 20-80 Hz) have been observed in the neocortex and hippocampus during behavioral arousal. Using computer simulations, we investigated the hypothesis that such rhythmic activity can emerge in a random network of interconnected GABAergic fast-spiking interneurons. Specific conditions for the population synchronization, on properties of single cells and the circuit, were identified. These include the following: (1) that the amplitude of spike afterhyperpolarization be above the GABAA synaptic reversal potential; (2) that the ratio between the synaptic decay time constant and the oscillation period be sufficiently large; (3) that the effects of heterogeneities be modest because of a steep frequency-current relationship of fast-spiking neurons. Furthermore, using a population coherence measure, based on coincident firings of neural pairs, it is demonstrated that large-scale network synchronization requires a critical (minimal) average number of synaptic contacts per cell, which is not sensitive to the network size. By changing the GABAA synaptic maximal conductance, synaptic decay time constant, or the mean external excitatory drive to the network, the neuronal firing frequencies were gradually and monotonically varied. By contrast, the network synchronization was found to be high only within a frequency band coinciding with the gamma (20-80 Hz) range. We conclude that the GABAA synaptic transmission provides a suitable mechanism for synchronized gamma oscillations in a sparsely connected network of fast-spiking interneurons. In turn, the interneuronal network can presumably maintain subthreshold oscillations in principal cell populations and serve to synchronize discharges of spatially distributed neurons. The presynaptic and postsynaptic cell have identical parameters and the variables in each cell are identified by using _pre or _post as a postfix to their names. The presynaptic cell influences the postsynaptic one via the synapse (variables and parameters: I_syn, E_syn, g_syn, F, theta_syn, alpha, beta). The applied current to the presynaptic cell, I_app_pre, is set to 2 microA/cm2 for 10 ms as in figure 1C of the article. The dependence of the postsynaptic cell on directly applied current can be investigated in isolation by setting I_app_pre to 0 and altering I_app_post. Originally created by libAntimony v1.4 (using libSBML 3.4.1)