ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Casler, J. C., Zajac, A. L., Valbuena, F. M., Sparvoli, D., Jeyifous, O., Turkewitz, A. P., Horne-Badovinac, S., Green, W. N., & Glick, B. S. ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms. Molecular Biology of the Cell, (2020): mbcE20090591, doi:10.1091/mbc.E20-09-0591.Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescentsecretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory proteinESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used withmany model organisms.This work was supported by NIH grant R01 GM104010 to BSG, by NIH grant R01 GM105783 to APT, by NIH grant R01 GM136961 and American Cancer Society grant RSG-14-176 to SHB, and by NIH grant R01 DA044760 to WNG. JCC was supported by NIH training grant T32 GM007183. AZ was supported by American Heart Association fellowship 16POST2726018 and American Cancer Society fellowship 132123-PF-18-025-01-CSM. Thanks for assistance with fluorescence microscopy to Vytas Bindokas and Christine Labno at the Integrated Microscopy Core Facility, which is supported by the NIH-funded Cancer Center Support Grant P30 CA014599. The pUASt-ManII-eGFP plasmid was a gift from Bing Ye, and the Ubi-Gal4 plasmid was a gift from Rick Fehon.2020-12-2

Microtubule plus-end binding protein CLASP2 in neural development

Normal brain function is dependent on the correct positioning and connectivity of neurons established during development. The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to lipoprotein receptors and inducing tyrosine phosphorylation of the intracellular adaptor protein Dab1. Mutations in genes of the Reelin signaling pathway lead to profound defects in neuronal positioning during brain development in both mice and humans. However, the molecular mechanisms by which Reelin controls neuronal morphology and migration are unknown. We have used a systems analysis approach to identify genes perturbed in the Reelin signaling pathway and identified microtubule stabilizing CLIP-associated protein 2 (CLASP2) as a key cytoskeletal modifier of Reelin mutant phenotypes. Currently, little is known about the role of CLASP2 in the developing brain. We propose that CLASP2 is a key effector in the Reelin signaling pathway controlling basic aspects of cortical layering, neuronal morphology, and function. CLASP2 is a plus-end tracking protein and this localization places CLASP2 in a strategic position to control neurite outgrowth, directionality, and responsiveness to extracellular cues. Our results demonstrate that CLASP2 expression correlates with neurite length and synaptic activity in primary neuron cultures; however, the role of CLASP2 during brain development was unknown. In this dissertation, we have characterized the role of CLASP2 during cortical development by in utero electroporation of shRNA plasmids and found that silencing CLASP2 in migrating neurons leads to mislocalized cells at deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. In addition, we found that GSK3β-mediated phosphorylation of CLASP2 controls Dab1 binding and is required for regulating CLASP2 effects on neuron morphology and migration. This dissertation provides the first steps in gaining insight into how Reelin signaling affects cytoskeletal reorganization to regulate fundamental features of neuronal migration, positioning and morphogenesis

The role of the Golgi apparatus in neuronal polarity

ABSTRACT The Golgi apparatus has always been an interesting organelle of study because of its unique morphology as well as the critical roles it plays in cell biology. It is situated next to the endoplasmic reticulum and secreted proteins must pass through the Golgi vesicular pathway for modifications and targeting. In addition, the Golgi apparatus plays an essential role in establishing cellular polarity. Cell polarity refers to difference in orientation of cell structures spatially, and is involved in establishing functionality. The Golgi apparatus establishes cell polarity in various ways including orienting itself spatially, biasing vesicular trafficking within the cell, and most importantly through its role as a microtubule organizing center. The cytoskeleton provides the structural framework for cells. Microtubules nucleated from the Golgi-dependent microtubule organizing center result in an asymmetric cytoskeleton. An asymmetric cytoskeleton is essential to establishing cell polarity. Neurons require cell polarity to establish the essential structures such as the axon and dendrites. The Golgi apparatus establishes neuronal polarity through its extensive network of associated proteins. In this review, we will discuss the growing evidence supporting the role of the Golgi apparatus in establishing neuronal polarity

Region-specific dendritic simplification induced by Aβ, mediated by tau via dysregulation of microtubule dynamics: a mechanistic distinct event from other neurodegenerative processes.

BackgroundDendritic simplification, a key feature of the neurodegenerative triad of Alzheimer's disease (AD) in addition to spine changes and neuron loss, occurs in a region-specific manner. However, it is unknown how changes in dendritic complexity are mediated and how they relate to spine changes and neuron loss.ResultsTo investigate the mechanisms of dendritic simplification in an authentic CNS environment we employed an ex vivo model, based on targeted expression of enhanced green fluorescent protein (EGFP)-tagged constructs in organotypic hippocampal slices of mice. Algorithm-based 3D reconstruction of whole neuron morphology in different hippocampal regions was performed on slices from APPSDL-transgenic and control animals. We demonstrate that induction of dendritic simplification requires the combined action of amyloid beta (Aβ) and human tau. Simplification is restricted to principal neurons of the CA1 region, recapitulating the region specificity in AD patients, and occurs at sites of Schaffer collateral input. We report that γ-secretase inhibition and treatment with the NMDA-receptor antagonist, CPP, counteract dendritic simplification. The microtubule-stabilizing drug epothilone D (EpoD) induces simplification in control cultures per se. Similar morphological changes were induced by a phosphoblocking tau construct, which also increases microtubule stability. In fact, low nanomolar concentrations of naturally secreted Aβ decreased phosphorylation at S262 in a cellular model, a site which is known to directly modulate tau-microtubule interactions.ConclusionsThe data provide evidence that dendritic simplification is mechanistically distinct from other neurodegenerative events and involves microtubule stabilization by dendritic tau, which becomes dephosphorylated at certain sites. They imply that treatments leading to an overall decrease of tau phosphorylation might have a negative impact on neuronal connectivity

J Cell Physiol

Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.R01 DK111949/DK/NIDDK NIH HHS/United StatesR01 DK095811/DK/NIDDK NIH HHS/United StatesH75 IP000287/IP/NCIRD CDC HHS/United StatesP30 DK058404/DK/NIDDK NIH HHS/United StatesR01 GM078373/GM/NIGMS NIH HHS/United StatesR01 DK106228/DK/NIDDK NIH HHS/United StatesR01 DK075555/DK/NIDDK NIH HHS/United States2019-02-01T00:00:00Z28548701PMC5673559vault:2511

Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity.

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions

A distinct Golgi-targeting mechanism of dGM130 in Drosophila neurons

GM130 is a matrix protein that is conserved in metazoans and involved in the architecture of the Golgi apparatus. In neurons, Golgi apparatus and dendritic Golgi outposts (GOs) have different compartmental organizations, and GM130 localization is present in both, indicating that GM130 has a unique Golgi-targeting mechanism. Here, we investigated the Golgi-targeting mechanism of the GM130 homologue, dGM130, using in vivo imaging of Drosophila dendritic arborization (da) neurons. The results showed that two independent Golgi-targeting domains (GTDs) with different Golgi localization characteristics in dGM130, together determined the precise localization of dGM130 in both the soma and dendrites. GTD1, covering the first coiled-coil region, preferentially targeted to somal Golgi rather than GOs; whereas GTD2, containing the second coiled-coil region and C-terminus, dynamically targeted to Golgi in both soma and dendrites. These findings suggest that there are two distinct mechanisms by which dGM130 targets to the Golgi apparatus and GOs, underlying the structural differences between them, and further provides new insights into the formation of neuronal polarity

Augmin complex components control branching of sensory neuron dendrites in <em>Drosophila</em> larvae

Microtubule is the major architectural element to support proper neuronal structure. It is tightly organized with intrinsic polarity and affects not only neuronal morphology but also the transport property within the cell. In many cell types, the centrosome component γ-tubulin is the principal microtubule nucleator. However, the mechanism underlying neuronal microtubule nucleation and organization remains unknown. During neuronal development, the centrosome is inactivated and microtubule nucleation becomes acentrosomal. Whether the microtubule centrosomal-independent nucleation contributes to the establishment of polarity in neurons remains unclear and essential to answer. The purpose of this work is to reveal whether Augmin mediated microtubule nucleation plays a role in building up proper dendritic morphology and organizing dendritic microtubule polarity. To this purpose, I analyzed the dendrite morphology of class IV ddaC da neurons in Drosophila larvae carrying mutations for γ-tubulin and Augmin. I found that dendritic morphology and dendrite branch dynamics were changed in γ-tubulin, dgt5, dgt6 (Augmin) and Dgp71WD (γ-TuRC) mutants. Interestingly, the phenotypes of these various mutants were similar, suggesting the possibility that they might act in concert. To test this possibility, I performed genetic interaction experiments between γtub23C, dgt5, dgt6 and Dgp71WD and found these molecules play coordinate roles in dendrite morphology. In Augmin complex mutant neurons, the localization of fluorescently tagged α-tubulin and the microtubule minusend marker Nod were both altered, suggesting a role of Augmin in microtubule organization in these neurons. Taken together, my work suggests a role of the Augmin complex in the proper organization of microtubules in neuronal dendrites, which is important for achieving dendritic complexity in Drosophila PNS class IV da neuron