Name: Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis (Video) | JoVE Journal
Uploaded: 2018-08-07T15:00:00.000+00:00
Duration: 8 min 55 s
Description: Learn live-cell microscopy and NMR techniques to characterize MLKL-mediated plasma membrane rupture in necroptosis using mouse embryonic fibroblasts. These methods directly implicate MLKL as the executioner of cell death.

Identifying genetic components of necroptosis has facilitated the use of animal models to test the implication of necroptosis in physiology and disease¹^,²^,³^,⁴^,⁵. Knockout of RIPK3 or MLKL in mice had minimal implication in development and adult homeostasis suggesting that necroptosis is not essential for life³^,⁶. Moreover, certain species do not contain either RIPK3 or MLKL genes, supporting the non-essential role of necroptosis in animals⁷^,⁸. On the other hand, challenging knockout animal models with various pathologies induced in the laboratory has revealed an important role of necroptosis in inflammation, innate immunity, and viral infection⁹^,¹⁰^,¹¹^,¹².

Necroptosis can be activated in several ways by signaling through different innate immunity sensors, all of which result in the activation of RIPK3¹^,¹³^,¹⁴. Active RIPK3 in turn phosphorylates and activates MLKL³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸. The most studied, and perhaps the most complex way, leading to activation of RIPK3 involves death receptor ligation, which bifurcates based on the downstream composition of the signaling complexes to either induce apoptosis or necroptosis¹. Necroptosis ensues when signaling through RIPK1 is favored and results in engagement of RIPK3¹⁹^,²⁰. This outcome is easily favored upon pharmacological inhibition or genetic deletion of caspase 8, a putative endogenous inhibitor of necroptosis that keeps necroptosis at bay. RIPK1 binds to and activates RIPK3. Another way to activate necroptosis is through Toll-like receptors TLR3/TLR4 signaling, which engages and activates RIPK3 through TIR-domain-containing adapter-inducing interferon-β (TRIF)²¹. Yet another way to die by necroptosis is by activation of the DNA sensor DAI, which directly engages and activates RIPK3²².

MLKL is a cytosolic protein comprised of an N-terminal helix bundle (NB) domain and a C-terminal pseudokinase domain (psKD) linked by a regulatory brace region³. In normal cells, MLKL is found in the cytosol where it is thought to be in an inactive complex with RIPK3¹⁴. Activation of necroptosis triggers RIPK3 phosphorylation of MLKL in the activation loop of the psKD, and potentially additional sites in the NB and brace³^,¹⁵^,²³. Phosphorylation induces a conformational change in MLKL that results in dissociation from RIPK3¹⁴. Poorly understood conformational changes release the brace from the psKD²⁴. The brace, which contains 2 helices, mediates oligomerization of MLKL into a putative trimer through the C-terminal helix²⁵. The N-terminal helix of the brace inhibits the NB domain, which is essential for membrane permeabilization²⁴^,²⁶. In isolation, NB domain is sufficient to induce plasma membrane permeabilization and necroptosis¹⁶^,²⁴^,²⁷. The pro-necroptotic activity of NB was reconstituted in mouse embryonic fibroblasts deficient in MLKL (mlkl^-/- MEFs). NB is a lipid binding domain that preferentially engages the phospholipid phosphatidylinositol 4,5 diphosphate (PIP₂). We proposed a stepwise mechanism of activation of MLKL, wherein brace oligomerization facilitates recruitment of MLKL to the plasma membrane via weak interactions of NB with the PIP₂ polar head group²⁴. At the membrane, the NB undergoes regulated exposure of an additional high-affinity binding site for PIP₂, which is masked by the brace in inactive MLKL. Overall, the multiple interactions of NB with PIP₂ destabilize the plasma membrane leading to its rupture, although the molecular mechanism of these events have not been elucidated.

Here we illustrate specific methods used to characterize the function of MLKL as executioner of necroptosis²⁴. In particular, we focus on the most minimal domain of MLKL, the NB and brace (NBB), which is regulated by brace inhibition and can be activated through enforced dimerization to induce plasma membrane rupture and necroptosis. We describe our inducible expression system combined with enforced drug-induced FKBP-mediated dimerization for live-cell imaging, and electron microscopy of cells undergoing necroptosis. Additionally, we illustrate our in vitro NMR analysis of the interactions of NBB with phosphatidylinositols (PIPs).