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1.
Biomed Opt Express ; 10(1): 29-49, 2019 Jan 01.
Article in English | MEDLINE | ID: mdl-30775081

ABSTRACT

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

2.
ISRN Cell Biol ; 20132013.
Article in English | MEDLINE | ID: mdl-25419467

ABSTRACT

Mitochondria, normally tubular and distributed throughout the cell, are instead found in spermatocytes in perinuclear clusters in close association with nuage, an amorphous organelle composed of RNA and RNA-processing proteins that generate piRNAs. piRNAs are a form of RNAi required for transposon suppression and ultimately fertility. MitoPLD, another protein required for piRNA production, is anchored to the mitochondrial surface, suggesting that the nuage, also known as intermitochondrial cement, needs to be juxtaposed there to bring MitoPLD into proximity with the remainder of the piRNA-generating machinery. However, the mechanism underlying the juxtaposition is unknown. Gasz, a multidomain protein of known function found in the nuage in vertebrates, is required for piRNA production and interacts with other nuage proteins involved in this pathway. Unexpectedly, we observed that Gasz, in nonspermatogenic mammalian cells lines, localizes to mitochondria and does so through a previously unrecognized conserved C-terminal mitochondrial targeting sequence. Moreover, in this setting, Gasz is able to recruit some of the normally nuage-localized proteins to the mitochondrial surface. Taken together, these findings suggest that Gasz is a nuage-localized protein in spermatocytes that facilitates anchoring of the nuage to the mitochondrial surface where piRNA generation takes place as a collaboration between nuage and mitochondrial-surface proteins.

3.
BMC Dev Biol ; 6: 60, 2006 Dec 07.
Article in English | MEDLINE | ID: mdl-17156430

ABSTRACT

BACKGROUND: Cellularization of the Drosophila embryo is an unusually synchronous form of cytokinesis in which polarized membrane extension proceeds in part through incorporation of new membrane via fusion of apically-translocated Golgi-derived vesicles. RESULTS: We describe here involvement of the signaling enzyme Phospholipase D (Pld) in regulation of this developmental step. Functional analysis using gene targeting revealed that cellularization is hindered by the loss of Pld, resulting frequently in early embryonic developmental arrest. Mechanistically, chronic Pld deficiency causes abnormal Golgi structure and secretory vesicle trafficking. CONCLUSION: Our results suggest that Pld functions to promote trafficking of Golgi-derived fusion-competent vesicles during cellularization.


Subject(s)
Cytokinesis , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Phospholipase D/physiology , Animals , COS Cells , Cell Line , Chemokine CX3CL1 , Chemokines, CX3C/analysis , Chlorocebus aethiops , Cytoplasmic Vesicles/chemistry , Cytoplasmic Vesicles/enzymology , Cytoplasmic Vesicles/physiology , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Enzyme Activation , Female , Golgi Apparatus/physiology , Humans , Male , Membrane Proteins/analysis , Membranes/metabolism , Mutation , Phospholipase D/genetics , RNA, Messenger, Stored/metabolism , Signal Transduction , Transfection
4.
Mol Biol Cell ; 16(6): 2614-23, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15772157

ABSTRACT

Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion.


Subject(s)
Insulin/pharmacology , Membrane Fusion/physiology , Phospholipase D/metabolism , Transport Vesicles/metabolism , 3T3-L1 Cells , Adenoviridae/genetics , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Biological Transport, Active , Blotting, Western , Deoxyglucose/metabolism , Enzyme Activation , Green Fluorescent Proteins/metabolism , Insulin/metabolism , Mice , Phosphatidic Acids/biosynthesis , Phospholipase D/analysis , RNA, Small Interfering/metabolism , Retroviridae/genetics
5.
J Cell Biol ; 162(2): 305-15, 2003 Jul 21.
Article in English | MEDLINE | ID: mdl-12876278

ABSTRACT

The signaling enzyme phospholipase D1 (PLD1) facilitates membrane vesicle trafficking. Here, we explore how PLD1 subcellular localization is regulated via Phox homology (PX) and pleckstrin homology (PH) domains and a PI4,5P2-binding site critical for its activation. PLD1 localized to perinuclear endosomes and Golgi in COS-7 cells, but on cellular stimulation, translocated to the plasma membrane in an activity-facilitated manner and then returned to the endosomes. The PI4,5P2-interacting site sufficed to mediate outward translocation and association with the plasma membrane. However, in the absence of PX and PH domains, PLD1 was unable to return efficiently to the endosomes. The PX and PH domains appear to facilitate internalization at different steps. The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization. In contrast, the PX domain appears to mediate binding to PI5P, a lipid newly recognized to accumulate in endocytosing vesicles. Finally, we show that the PH domain-dependent translocation step, but not the PX domain, is required for PLD1 to function in regulated exocytosis in PC12 cells. We propose that PLD1 localization and function involves regulated and continual cycling through a succession of subcellular sites, mediated by successive combinations of membrane association interactions.


Subject(s)
Cell Membrane/enzymology , Endosomes/enzymology , Gene Expression Regulation, Enzymologic , Golgi Apparatus/enzymology , Membrane Microdomains/enzymology , Phospholipase D/metabolism , Amino Acid Motifs , Animals , COS Cells , Cell Membrane/metabolism , Chlorocebus aethiops , Endosomes/metabolism , Enzyme Activation , Exocytosis , Golgi Apparatus/metabolism , Membrane Microdomains/metabolism , Models, Biological , Mutagenesis, Site-Directed , PC12 Cells , Phosphatidylinositol Phosphates/metabolism , Phospholipase D/chemistry , Phospholipase D/genetics , Rats , Subcellular Fractions/enzymology
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