In vitro reconstitution demonstrates the amyloid-beta mediated myelin membrane deformation

Amyloid-beta (Ab) aggregation mediated neuronal membrane deformation, although poorly understood, is implicated in Alzheimer's Disease (AD). Particularly, whether Ab aggregation can induce neuronal demyelination remains unknown. Here we show that Aβ-40 binds and induces extensive tubulation in the myelin membrane in vitro. The binding of Aβ-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin membrane in the early time scales. Furthermore, elongation of Aβ-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the myelin membrane followed by extensive membrane tubulation observed in the late phase. Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of Aβ-40- myelin membrane interaction and demonstrate how short timescale, local phenomena of binding, and fibril mediated load generation manifests into long timescale, global phenomena of myelin tubulation and demonstrates the ability of Aβ-40 to demyelinate.

MICAL-L1 is required for cargo protein delivery to the cell surface

ABSTRACT Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signaling, immunity, neuroprotection, and virus infection. The retromer core (VPS26/VPS29/VPS35) is present on cargo-transporting, tubular carriers along with a range of sorting nexins. Here, we elucidate the structural basis of membrane tubulation and coupled cargo recognition by metazoan and fungal retromer coats assembled with the non–Bin1/Amphiphysin/Rvs (BAR) sorting nexin SNX3 using cryo–electron tomography. The retromer core retains its arched, scaffolding structure but changes its mode of membrane recruitment when assembled with different SNX adaptors, allowing cargo recognition at subunit interfaces. Thus, membrane bending and cargo incorporation can be modulated to allow retromer to traffic cargoes along different cellular transport routes.

KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil–coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

FIB/SEM-based analysis of Borrelia intracellular processing by human macrophages

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells like macrophages is decisive for the outcome of a respective infection. Here, we use for the first time focused ion beam scanning electron microscopy tomography (FIB/SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction, but hard to visualize in live cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2

ABSTRACTThe membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.

Regulation of caveolae through cholesterol-depletion dependent tubulation by PACSIN2/Syndapin II

The membrane shaping ability of PACSIN2 via its FCH-BAR (F-BAR) domain has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membrane contains abundant levels of cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. We show that the binding of PACSIN2 to the membrane could be negatively regulated by the amount of cholesterol in the membrane. We prepared a reconstituted membrane based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity to the F-BAR domain of PACSIN2 than the membrane without cholesterol, presumably due to a decrease in electrostatic charge density. Consistently, the acute depletion of cholesterol from the plasma membrane resulted in the appearance of PACSIN2-localized tubules with caveolin-1 at their tips, suggesting that the presence of cholesterol inhibited the prominent membrane tubulation by PACSIN2. The tubules induced by PACSIN2 were suggested to be an intermediate of caveolae endocytosis. Consistently, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the cells deficient in PACSIN2. These data suggested that PACSIN2 mediated the caveolae internalization dependently on the amount of cholesterol at the plasma membrane, providing a possible mechanism for the cholesterol-dependent regulation of caveolae.