Kinesin-II motors differentially impact biogenesis of distinct extracellular vesicle subpopulations shed from C. elegans sensory cilia

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium, a conserved signaling organelle. The mechanisms underlying cargo enrichment and biogenesis of heterogeneous EVs shed from cilia are unclear. Here we discover the conserved ion channel CLHM-1 as a new ciliary EV cargo. Using super-resolution microscopy, we imaged EVs released into the environment from sensory neuron cilia of C. elegans expressing fluorescently-tagged CLHM-1 and TRP polycystin-2 channel PKD-2 EV cargoes at endogenous levels. We find that these proteins are enriched in distinct EV subpopulations that are differentially shed in response to availability of hermaphrodite mating partners. Both CLHM-1 and PKD-2 localize to the ciliary base and middle segment of the cilium proper, but PKD-2 alone is present in the cilium distal tip and EVs shed from this site. CLHM-1 EVs released into the environment bud from a secondary site, the periciliary membrane compartment at the ciliary base. We show that individual heterotrimeric and homomeric kinesin-II motors have discrete impacts on the colocalization of PKD-2 and CLHM-1 in both cilia and EVs. Total loss of kinesin-II activity significantly decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde kinesin-II-dependent intraflagellar transport is required for selective enrichment of specific protein cargoes into heterogeneous EVs with different signaling potentials.

The Salmonella effector SifA initiates a kinesin-1 and kinesin-3 recruitment process mirroring that mediated by Arl8a/b

When intracellular, pathogenic Salmonella reside in a membrane compartment composed of interconnected vacuoles and tubules, the formation of which depends on the translocation of bacterial effectors into the host cell. Cytoskeletons and their molecular motors are prime targets for these effectors. In this study, we show that the microtubule molecular motor KIF1Bß, a member of the kinesin-3 family, is a key element for the establishment of the Salmonella replication niche as its absence is detrimental to the stability of bacterial vacuoles and the formation of associated tubules. Kinesin-3 interacts with the Salmonella effector SifA but also with SKIP, a host protein complexed to SifA. The interaction with SifA is essential for the recruitment of kinesin-3 on Salmonella vacuoles while that with SKIP is incidental. In the non-infectious context, however, the interaction with SKIP is essential for the recruitment and activity of kinesin-3 on a part of lysosomes. Finally, our results show that in infected cells, the presence of SifA establishes a kinesin-1 and kinesin-3 recruitment pathway that is analogous to and functions independently of that mediated by the Arl8a/b GTPases.

The Role of PDE11A4 in Social Isolation-Induced Changes in Intracellular Signaling and Neuroinflammation

Phosphodiesterase 11A (PDE11A), an enzyme that degrades cyclic nucleotides (cAMP and cGMP), is the only PDE whose mRNA expression in brain is restricted to the hippocampal formation. Previously, we showed that chronic social isolation changes subsequent social behaviors in adult mice by reducing expression of PDE11A4 in the membrane fraction of the ventral hippocampus (VHIPP). Here we seek extend these findings by determining 1) if isolation-induced decreases in PDE11A4 require chronic social isolation or if they occur acutely and are sustained long-term, 2) if isolation-induced decreases occur uniquely in adults (i.e., not adolescents), and 3) how the loss of PDE11 signaling may increase neuroinflammation. Both acute and chronic social isolation decrease PDE11A4 expression in adult but not adolescent mice. This decrease in PDE11A4 is specific to the membrane compartment of the VHIPP, as it occurs neither in the soluble nor nuclear fractions of the VHIPP nor in any compartment of the dorsal HIPP. The effect of social isolation on membrane PDE11A4 is also selective in that PDE2A and PDE10A expression remain unchanged. Isolation-induced decreases in PDE11A4 expression appear to be functional as social isolation elicited changes in PDE11A-relevant signal transduction cascades (i.e., decreased pCamKIIα and pS6-235/236) and behavior (i.e., increased remote long-term memory for social odor recognition). Interestingly, we found that isolation-induced decreases in membrane PDE11A4 correlated with increased expression of interleukin-6 (IL-6) in the soluble fraction, suggesting pro-inflammatory signaling for this cytokine. This effect on IL-6 is consistent with the fact that PDE11A deletion increased microglia activation, although it left astrocytes unchanged. Together, these data suggest that isolation-induced decreases in PDE11A4 may alter subsequent social behavior via increased neuroinflammatory processes in adult mice.

Ectocytosis prevents accumulation of ciliary cargo in C. elegans sensory neurons

Cilia are sensory organelles protruding from cell surfaces. Release of extracellular vesicles (EVs) from cilia was previously observed in mammals, Chlamydomonas, and in male Caenorhabditis elegans. Using the EV marker TSP-6 (an ortholog of mammalian CD9) and other ciliary receptors, we show that EVs are formed from ciliated sensory neurons in C. elegans hermaphrodites. Release of EVs is observed from two ciliary locations: the cilia tip and/or periciliary membrane compartment (PCMC). Outward budding of EVs from the cilia tip leads to their release into the environment. EVs’ budding from the PCMC is concomitantly phagocytosed by the associated glial cells. To maintain cilia composition, a tight regulation of cargo import and removal is achieved by the action of intra-flagellar transport (IFT). Unbalanced IFT due to cargo overexpression or mutations in the IFT machinery leads to local accumulation of ciliary proteins. Disposal of excess ciliary proteins via EVs reduces their local accumulation and exports them to the environment and/or to the glia associated to these ciliated neurons. We suggest that EV budding from cilia subcompartments acts as a safeguard mechanism to remove deleterious excess of ciliary material.

Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

Modulation of Temoporfin Distribution in Blood by β-Cyclodextrin Nanoshuttles

Photodynamic therapy represents a more targeted and less invasive alternative cancer treatment to traditional modalities. Temoporfin, as with many photosensitizers, is given by injection into a vein, and its subsequent fate is largely determined by the binding to plasma proteins and interaction with endothelial and blood cells. Thus, it is essential to be able to control and to alter the biodistribution of temoporfin in blood. In the present study, we evaluated the effect of co-administration of temoporfin with randomly methylated β-CD (Me-β-CD) on the distribution of temoporfin in the main subpopulations of blood cells of healthy donors using absorbance spectrophotometry and flow cytometry. We showed that cell-bound temoporfin fraction in blood strongly depends on the concentration of Me-β-CD. In fact, the accumulation of temoporfin in white blood cells was more sensitive than that in red blood cells, due to the higher volume of membranous organelles in white blood cells. Finally, we demonstrated that Me-β-CD significantly increases cellular uptake of temoporfin cancer human Burkitt′s lymphoma Raji cells. The presence of Me-β-CD resulted in a spotted pattern of temoporfin distribution in the plasma membrane compartment. Our results clearly demonstrated that β-CDs derivatives provide new options to modulate temoporfin biodistribution in blood.

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip 1

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveal distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tri-cellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function during junction formation and stabilization during sprouting angiogenesis.

Molecular Imaging with Genetically Programmed Nanoparticles

Nanoparticle research has greatly benefitted medical imaging platforms by generating new signals, enhancing detection sensitivity, and expanding both clinical and preclinical applications. For magnetic resonance imaging, the fabrication of superparamagnetic iron oxide nanoparticles has provided a means of detecting cells and has paved the way for magnetic particle imaging. As the field of molecular imaging grows and enables the tracking of cells and their molecular activities so does the possibility of tracking genetically programmed biomarkers. This chapter discusses the advantages and challenges of gene-based contrast, using the bacterial magnetosome model to highlight the requirements of in vivo iron biomineralization and reporter gene expression for magnetic resonance signal detection. New information about magnetosome protein interactions in non-magnetic mammalian cells is considered in the light of design and application(s) of a rudimentary magnetosome-like nanoparticle for molecular imaging. Central to this is the hypothesis that a magnetosome root structure is defined by essential magnetosome genes, whose expression positions the biomineral in a given membrane compartment, in any cell type. The use of synthetic biology for programming multi-component structures not only broadens the scope of reporter gene expression for molecular MRI but also facilitates the tracking of cell therapies.

In vivo proximity labeling identifies cardiomyocyte protein networks during zebrafish heart regeneration

Strategies have not been available until recently to uncover interacting protein networks specific to key cell types, their subcellular compartments, and their major regulators during complex in vivo events. Here, we apply BioID2 proximity labeling to capture protein networks acting within cardiomyocytes during a key model of innate heart regeneration in zebrafish. Transgenic zebrafish expressing a promiscuous BirA2 localized to the entire myocardial cell or membrane compartment were generated, each identifying distinct proteomes in adult cardiomyocytes that became altered during regeneration. BioID2 profiling for interactors with ErbB2, a co-receptor for the cardiomyocyte mitogen Nrg1, implicated Rho A as a target of ErbB2 signaling in cardiomyocytes. Blockade of Rho A during heart regeneration, or during cardiogenic stimulation by the mitogenic influences Nrg1, Vegfaa, or vitamin D, disrupted muscle creation. Our findings reveal proximity labeling as a useful resource to interrogate cell proteomes and signaling networks during tissue regeneration in zebrafish.