Karen A. Newell-Litwa

Neuronal and non-neuronal functions of the AP-3 sorting machinery.

Vesicles selectively exchange lipids, membrane proteins and luminal contents between organelles along the exocytic and endocytic routes. The repertoire of membrane proteins present in these vesicles is crucial for their targeting and function. Vesicle composition is determined at the time of their biogenesis by cytosolic coats. The heterotetrameric protein adaptor protein complex 3 (AP-3), a coat component, participates in the generation of a diverse group of secretory organelles and lysosome-related organelles. Recent work has shed light on the mechanisms that regulate AP-3 and the trafficking pathways controlled by this adaptor. Phenotypic analysis of organisms carrying genetic deficiencies in the AP-3 pathway highlight its role regulating the targeting of lysosomal, melanosomal and synaptic vesicle-specific membrane proteins. Synaptic vesicles from AP-3-deficient mice possess altered levels of neurotransmitter and ion transporters, molecules that ultimately define the type and amount of neurotransmitter stored in these vesicles. These findings reveal a complex picture of how AP-3 functions in multiple tissues, including neuronal tissue, and expose potential links between endocytic sorting mechanisms and the pathogenesis of psychiatric disorders such as schizophrenia.

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front–back polarity in migrating cells

Myosin IIA and IIB synergistically generate front–back polarity through their effects on actomyosin bundling formation and stability, and adhesion maturation, which are mediated by localized Rac GEF depletion.

Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky-Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1(-/-)) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2(-/-)), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes.

Myosin IIB Activity and Phosphorylation Status Determines Dendritic Spine and Post-Synaptic Density Morphology

Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD) and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB) activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in mushroom-shaped spines with an enlarged PSD.

Hermansky-Pudlak protein complexes, AP-3 and BLOC-1, differentially regulate presynaptic composition in the striatum and hippocampus.

Endosomal sorting mechanisms mediated by AP-3 and BLOC-1 are perturbed in Hermansky-Pudlak Syndrome, a human genetic condition characterized by albinism and prolonged bleeding (OMIM #203300). Additionally, mouse models defective in either one of these complexes possess defective synaptic vesicle biogenesis (Newell-Litwa et al., 2009). These synaptic vesicle phenotypes were presumed uniform throughout the brain. However, here we report that AP-3 and BLOC-1 differentially regulate the composition of presynaptic terminals in the striatum and dentate gyrus of the hippocampus. Quantitative immunoelectron microscopy demonstrated that the majority of AP-3 immunoreactivity in both wild-type striatum and hippocampus localizes to presynaptic axonal compartments, where it regulates synaptic vesicle size. In the striatum, loss of AP-3 (Ap3dmh/mh ) resulted in decreased synaptic vesicle size. In contrast, loss of AP-3 in the dentate gyrus increased synaptic vesicle size, thus suggesting anatomically specific AP-3-regulatory mechanisms. Loss-of-function alleles of BLOC-1, Pldnpa/pa , and Mutedmu/mu revealed that this complex acts as a brain-region-specific regulator of AP-3. In fact, BLOC-1 deficiencies selectively reduced AP-3 and AP-3 cargo immunoreactivity in presynaptic compartments within the dentate gyrus both at the light and/or electron microscopy level. However, the striatum did not exhibit these BLOC-1-null phenotypes. Our results demonstrate that distinct brain regions differentially regulate AP-3-dependent synaptic vesicle biogenesis. We propose that anatomically restricted mechanisms within the brain diversify the biogenesis and composition of synaptic vesicles.

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity.

ROCK1 forms the stable actomyosin filament bundles that initiate front–back and dendritic spine polarity, while ROCK2 regulates contractile force, Rac1 activity, and cofilin-mediated actin remodeling at the leading edge of migratory cells and the spine head of neurons.

The subcellular localization of the Niemann-Pick Type C proteins depends on the adaptor complex AP-3

Niemann-Pick Type C (NP-C) disease, caused by mutations in either human NPC1 (hNPC1) or human NPC2 (hNPC2), is characterized by the accumulation of unesterified cholesterol in late endosomes. Although it is known that the NP-C proteins are targeted to late endosomal/lysosomal compartments, their delivery mechanisms have not been fully elucidated. To identify mechanisms regulating NP-C protein localization, we used Saccharomyces cerevisiae, which expresses functional homologs of both NP-C proteins – scNcr1p and scNpc2p. Targeting of scNcr1p to the vacuole was perturbed in AP-3-deficient yeast cells, whereas the delivery of scNpc2p was affected by deficiencies in either AP-3 or GGA. We focused on the role of the AP-3 pathway in the targeting of the mammalian NP-C proteins. We found that, although mouse NPC1 (mNPC1) and hNPC2 co-localize with AP-3 to a similar extent in fibroblasts, hNPC2 preferentially co-localizes with AP-1. Importantly, the targeting of both mammalian NPC1 and NPC2 is dependent on AP-3. Moreover, and consistent with the NP-C proteins playing a role in cholesterol metabolism, AP-3-deficient cells have reduced levels of cholesterol. These results provide information about how the NP-C proteins are targeted to their sites of action and illustrate the possibility that defective sorting of the NP-C proteins along the endocytic route can alter cellular cholesterol.

Non-muscle myosin II in disease: mechanisms and therapeutic opportunities

ABSTRACT The actin motor protein non-muscle myosin II (NMII) acts as a master regulator of cell morphology, with a role in several essential cellular processes, including cell migration and post-synaptic dendritic spine plasticity in neurons. NMII also generates forces that alter biochemical signaling, by driving changes in interactions between actin-associated proteins that can ultimately regulate gene transcription. In addition to its roles in normal cellular physiology, NMII has recently emerged as a critical regulator of diverse, genetically complex diseases, including neuronal disorders, cancers and vascular disease. In the context of these disorders, NMII regulatory pathways can be directly mutated or indirectly altered by disease-causing mutations. NMII regulatory pathway genes are also increasingly found in disease-associated copy-number variants, particularly in neuronal disorders such as autism and schizophrenia. Furthermore, manipulation of NMII-mediated contractility regulates stem cell pluripotency and differentiation, thus highlighting the key role of NMII-based pharmaceuticals in the clinical success of stem cell therapies. In this Review, we discuss the emerging role of NMII activity and its regulation by kinases and microRNAs in the pathogenesis and prognosis of a diverse range of diseases, including neuronal disorders, cancer and vascular disease. We also address promising clinical applications and limitations of NMII-based inhibitors in the treatment of these diseases and the development of stem-cell-based therapies. Summary: Non-muscle myosin II (NMII) contributes to diverse diseases, including neuronal disorders, cancer and vascular disease. We address the role of NMII in disease pathology and highlight promising clinical applications of NMII inhibitors for disease and stem-cell-based therapies.

Cell migration: PKA and RhoA set the pace.

A new study shows that protein kinase A (PKA) activity establishes a signaling loop that governs protrusion-retraction cycles in migrating cells. PKA activity near the leading edge of protrusions phosphorylates RhoA and inhibits its activity via increased association with RhoGDI.

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later maturation. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine precursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synapses. Our observations demonstrate that specific combinations of RhoGTPase regulatory proteins temporally balance RhoGTPase activity during post-synaptic spine development.