Lysosome-related organelles contain an expansion compartment that mediates delivery of zinc transporters to promote homeostasis

Lysosome-related organelles play evolutionarily conserved roles in zinc storage, but mechanisms that control zinc flow in and out are not well understood. In C. elegans intestinal cells, the CDF-2 transporter stores zinc in these organelles during excess. Here we identify ZIPT-2.3 as the transporter that releases zinc during deficiency. The expression levels of CDF-2 and ZIPT-2.3 are reciprocally regulated in zinc excess and deficiency, establishing a fundamental mechanism of homeostasis. Super-resolution microscopy demonstrated these organelles are composed of a spherical acidified compartment and a hemispherical expansion compartment. The expansion compartment inflates during zinc excess and deficiency by vesicle fusion delivering zinc transporters. These results identify an unexpected structural feature of lysosome-related organelles that facilitates rapid transitions in the composition of zinc transporters to mediate homeostasis.

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Lysosome-Related Organelles in Intestinal Cells Are a Zinc Storage Site in C. elegans

Cell Metabolism ◽

10.1016/j.cmet.2011.12.003 ◽

2012 ◽

Vol 15 (1) ◽

pp. 88-99 ◽

Cited By ~ 75

Author(s):

Hyun Cheol Roh ◽

Sara Collier ◽

James Guthrie ◽

J. David Robertson ◽

Kerry Kornfeld

Keyword(s):

Intestinal Cells ◽

Storage Site ◽

C Elegans ◽

Lysosome Related Organelles ◽

Zinc Storage

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Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011352 ◽

2019 ◽

Vol 295 (3) ◽

pp. 729-742

Author(s):

Kieu T. M. Pham ◽

Ziyin Li

Keyword(s):

Trypanosoma Brucei ◽

Basal Body ◽

Super Resolution ◽

Specific Protein ◽

Protein Stabilization ◽

Structured Illumination ◽

Human Parasite ◽

Evolutionarily Conserved ◽

Body Components ◽

Super Resolution Microscopy

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei. Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

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Periodic actin structures in neuronal axons are required to maintain microtubules

10.1101/049379 ◽

2016 ◽

Cited By ~ 1

Author(s):

Yue Qu ◽

Ines Hahn ◽

Stephen Webb ◽

Simon P. Pearce ◽

Andreas Prokop

Keyword(s):

Super Resolution ◽

Close Correlation ◽

Membrane Skeleton ◽

Cortical Actin ◽

Evolutionarily Conserved ◽

Genes Encoding ◽

Neuronal Processes ◽

And Function ◽

Super Resolution Microscopy ◽

Actin Rings

SummaryAxons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal we observed gaps in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration.Significance statementAxons are cable-like neuronal processes that are up to a meter long in humans. These delicate structures often need to be maintained for an organism’s lifetime, i.e. up to a century in humans. Unsurprisingly, we gradually lose about 50% of axons as we age. Bundles of microtubules form the structural backbones and highways for life-sustaining transport within axons, and maintenance of these bundles is essential for axonal longevity. However, the mechanisms which actively maintain axonal microtubules are poorly understood. Here we identify cortical actin as an important factor maintaining microtubule polymerisation in axons. This finding provides potential explanations for the previously identified, but unexplained, links between mutations in genes encoding cortical actin regulators and neurodegeneration.

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Natural Viruses of Caenorhabditis Nematodes

Annual Review of Genetics ◽

10.1146/annurev-genet-112618-043756 ◽

2019 ◽

Vol 53 (1) ◽

pp. 313-326 ◽

Cited By ~ 4

Author(s):

Marie-Anne Félix ◽

David Wang

Keyword(s):

Model Organism ◽

Laboratory Model ◽

Intestinal Cells ◽

Caenorhabditis Briggsae ◽

C Elegans ◽

The Past ◽

Antiviral Genes ◽

Evolutionarily Conserved ◽

Positive Sense ◽

Orsay Virus

Caenorhabditis elegans has long been a laboratory model organism with no known natural pathogens. In the past ten years, however, natural viruses have been isolated from wild-caught C. elegans (Orsay virus) and its relative Caenorhabditis briggsae (Santeuil virus, Le Blanc virus, and Melnik virus). All are RNA positive-sense viruses related to Nodaviridae; they infect intestinal cells and are horizontally transmitted. The Orsay virus capsid structure has been determined and the virus can be reconstituted by transgenesis of the host. Recent use of the Orsay virus has enabled researchers to identify evolutionarily conserved proviral and antiviral genes that function in nematodes and mammals. These pathways include endocytosis through SID-3 and WASP; a uridylyltransferase that destabilizes viral RNAs by uridylation of their 3′ end; ubiquitin protein modifications and turnover; and the RNA interference pathway, which recognizes and degrades viral RNA.

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

10.1101/2021.12.19.473369 ◽

2021 ◽

Author(s):

Michael Clupper ◽

Rachael Gill ◽

Malek Elsayyid ◽

Denis Touroutine ◽

Jeffrey L. Caplan ◽

...

Keyword(s):

Super Resolution ◽

Intraflagellar Transport ◽

Extracellular Vesicle ◽

Specific Protein ◽

Secondary Site ◽

Selective Enrichment ◽

C Elegans ◽

Sensory Cilia ◽

Membrane Compartment ◽

Super Resolution Microscopy

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.

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Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽

10.32607/2075851-2017-9-4-42-51 ◽

2017 ◽

Vol 9 (4) ◽

pp. 42-51

Author(s):

S. S. Ryabichko ◽

◽

A. N. Ibragimov ◽

L. A. Lebedeva ◽

E. N. Kozlov ◽

...

Keyword(s):

Cell Nucleus ◽

Super Resolution ◽

Structure And Function ◽

And Function ◽

Super Resolution Microscopy

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Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

10.26434/chemrxiv.9209450.v1 ◽

2019 ◽

Author(s):

Jeffrey Chang ◽

Matthew Romei ◽

Steven Boxer

Keyword(s):

Rational Design ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Unit Cell Size ◽

Chlorine Substituent ◽

Gfp Chromophore ◽

The One ◽

Green Fluorescent ◽

Super Resolution Microscopy

Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

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