A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution.

Alzheimer's disease's established primary trigger is ß-amyloid (Aß) (Mucke and Selkoe, 2012). The amyloid precursor protein (APP) endocytosis is required for Aß generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes depends on its sorting for degradation in lysosomes ( Haass et al., 1992 ; Morel et al., 2013 ; Edgar et al., 2015 ; Ubelmann et al., 2017 ). The following endocytosis assay has been optimized to assess the amyloid precursor protein (APP) endocytosis and degradation by live murine cortical primary neurons ( Ubelmann et al., 2017 ).

Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells.

The established primary trigger of Alzheimer's disease's is ß-amyloid (Aß) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aß generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane ( Koo et al., 1996 ; Ubelmann et al., 2017 ). The following recycling assay has been optimized to assess APP recycling by live murine Neuro2a cells, a neuroblastoma cell line ( Ubelmann et al., 2017 ).

Bin1 and CD2AP polarise the endocytic generation of beta-amyloid.

The mechanisms driving pathological beta-amyloid (Aß) generation in late-onset Alzheimer's disease (AD) are unclear. Two late-onset AD risk factors, Bin1 and CD2AP, are regulators of endocytic trafficking, but it is unclear how their endocytic function regulates Aß generation in neurons. We identify a novel neuron-specific polarisation of Aß generation controlled by Bin1 and CD2AP We discover that Bin1 and CD2AP control Aß generation in axonal and dendritic early endosomes, respectively. Both Bin1 loss of function and CD2AP loss of function raise Aß generation by increasing APP and BACE1 convergence in early endosomes, however via distinct sorting events. When Bin1 levels are reduced, BACE1 is trapped in tubules of early endosomes and fails to recycle in axons. When CD2AP levels are reduced, APP is trapped at the limiting membrane of early endosomes and fails to be sorted for degradation in dendrites. Hence, Bin1 and CD2AP keep APP and BACE1 apart in early endosomes by distinct mechanisms in axon and dendrites. Individuals carrying variants of either factor would slowly accumulate Aß in neurons increasing the risk for late-onset AD.

Apical invasion of intestinal epithelial cells by Salmonella typhimurium requires villin to remodel the brush border actin cytoskeleton.

Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, leads to a decrease in Salmonella invasion. Villin is necessary for early membrane-associated processes and for optimal ruffle assembly by balancing the steady-state level of actin. The severing activity of villin is important for Salmonella invasion in vivo. The bacterial phosphatase SptP tightly regulates villin phosphorylation, while the actin-binding effector SipA protects F-actin and counterbalances villin-severing activity. Thus, villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of Salmonella entry.

Enterocyte loss of polarity and gut wound healing rely upon the F-actin-severing function of villin.

Efficient wound healing is required to maintain the integrity of the intestinal epithelial barrier because of its constant exposure to a large variety of environmental stresses. This process implies a partial cell depolarization and the acquisition of a motile phenotype that involves rearrangements of the actin cytoskeleton. Here we address how polarized enterocytes harboring actin-rich apical microvilli undergo extensive cell remodeling to drive injury repair. Using live imaging technologies, we demonstrate that enterocytes in vitro and in vivo rapidly depolarize their microvilli at the wound edge. Through its F-actin-severing activity, the microvillar actin-binding protein villin drives both apical microvilli disassembly in vitro and in vivo and promotes lamellipodial extension. Photoactivation experiments indicate that microvillar actin is mobilized at the lamellipodium, allowing optimal migration. Finally, efficient repair of colonic mechanical injuries requires villin severing of F-actin, emphasizing the importance of villin function in intestinal homeostasis. Thus, villin severs F-actin to ensure microvillus depolarization and enterocyte remodeling upon injury. This work highlights the importance of specialized apical pole disassembly for the repolarization of epithelial cells initiating migration.

A new role for the architecture of microvillar actin bundles in apical retention of membrane proteins.

Actin-bundling proteins are identified as key players in the morphogenesis of thin membrane protrusions. Until now, functional redundancy among the actin-bundling proteins villin, espin, and plastin-1 has prevented definitive conclusions regarding their role in intestinal microvilli. We report that triple knockout mice lacking these microvillar actin-bundling proteins suffer from growth delay but surprisingly still develop microvilli. However, the microvillar actin filaments are sparse and lack the characteristic organization of bundles. This correlates with a highly inefficient apical retention of enzymes and transporters that accumulate in subapical endocytic compartments. Myosin-1a, a motor involved in the anchorage of membrane proteins in microvilli, is also mislocalized. These findings illustrate, in vivo, a precise role for local actin filament architecture in the stabilization of apical cargoes into microvilli. Hence, the function of actin-bundling proteins is not to enable microvillar protrusion, as has been assumed, but to confer the appropriate actin organization for the apical retention of proteins essential for normal intestinal physiology.

Actin dynamics counteract membrane tension during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between 'actin dependent and 'actin independent'. We also find that light-chain-bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.