scholarly journals The dynamic Nexus: gap junctions control protein localization and mobility in distinct and surprising ways

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sean McCutcheon ◽  
Randy F. Stout ◽  
David C. Spray
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Protein Localization ◽  
Second Messengers ◽  
Ionic Currents ◽  
Cellular Interactions ◽  
Dynamic Membrane ◽  
Associated Proteins ◽  
The Stability ◽  
Membrane Organelle ◽  
Control Protein

Abstract Gap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Their function is critical for numerous cellular interactions, providing exchange of metabolites, signaling molecules, and ionic currents. GJ channels are composed of Connexin (Cx) hexamers paired across extracellular space and typically form large rafts of clustered channels, called plaques, at cell appositions. Cxs together with molecules that interact with GJ channels make up a supramolecular structure known as the GJ Nexus. While the stability of connexin localization in GJ plaques has been studied, mobility of other Nexus components has yet to be addressed. Colocalization analysis of several nexus components and other membrane proteins reveal that certain molecules are excluded from the GJ plaque (Aquaporin 4, EAAT2b), while others are quite penetrant (lipophilic molecules, Cx30, ZO-1, Occludin). Fluorescence recovery after photobleaching of tagged Nexus-associated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins. These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters.

scholarly journals The dynamic Nexus: Gap junctions control protein localization and mobility in distinct and surprising ways

2020 ◽  
Author(s):  
Sean McCutcheon ◽  
Randy F. Stout ◽  
David C. Spray
Keyword(s):  
Gap Junctions ◽  
Protein Localization ◽  
Second Messengers ◽  
Cellular Interactions ◽  
Dynamic Membrane ◽  
New Information ◽  
Associated Proteins ◽  
Summary Statement ◽  
The Stability ◽  
Membrane Organelle

AbstractGap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Their function is critical for numerous cellular interactions. GJ channels are composed of Connexin (Cx) hexamers paired across extracellular space and typically form large rafts of clustered channels, called plaques, at cell appositions. Cxs together with molecules that interact with GJ channels make up a supramolecular structure known as the GJ Nexus. While the stability of connexin localization in GJ plaques has been studied, mobility of other Nexus components has yet to be addressed. Colocalization analysis of several nexus components and other membrane proteins reveal that certain molecules are excluded from the GJ plaque (Aquaporin 4, EAAT2b), while others are quite penetrant (lipophilic molecules, Cx30, ZO-1, Occludin). Fluorescence recovery after photobleaching (FRAP) of tagged Nexus-associated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins. These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters.Summary StatementGap junctions are clustered membrane channels in plasma membrane of astrocytes and other cells. We report new information on how gap junctions control location and mobility of other astrocyte proteins.

scholarly journals The Putative RNA-Binding Protein Nrp1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered Into Heat-Induced Protein Aggregates in Fission Yeast

2021 ◽  
Author(s):  
Masashi Yukawa ◽  
Mitsuki Ohishi ◽  
Yusuke Yamada ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Aggregates ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
The Stability ◽  
And Function ◽  
Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here we show that deletion of the nrp1 gene, which encodes a putative RNA-binding protein with unknown function, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the nrp1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Nrp1 are essential for its cytoplasmic localization and function. We have also found that a portion of Nrp1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Nrp1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

scholarly journals Hypoxia elicits broad and systematic changes in protein subcellular localization

2011 ◽  
Vol 301 (4) ◽  
pp. C913-C928 ◽  
Author(s):  
Robert Michael Henke ◽  
Ranita Ghosh Dastidar ◽  
Ajit Shah ◽  
Daniela Cadinu ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Localization ◽  
Cell Periphery ◽  
Protein Distribution ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Regulatory ◽  
Chromatin Remodeling Complexes ◽  
Oxygen Signaling ◽  
Cellular Compartments ◽  
Control Protein

Oxygen provides a crucial energy source in eukaryotic cells. Hence, eukaryotes ranging from yeast to humans have developed sophisticated mechanisms to respond to changes in oxygen levels. Regulation of protein localization, like protein modifications, can be an effective mechanism to control protein function and activity. However, the contribution of protein localization in oxygen signaling has not been examined on a genomewide scale. Here, we examine how hypoxia affects protein distribution on a genomewide scale in the model eukaryote, the yeast Saccharomyces cerevisiae . We demonstrate, by live cell imaging, that hypoxia alters the cellular distribution of 203 proteins in yeast. These hypoxia-redistributed proteins include an array of proteins with important functions in various organelles. Many of them are nuclear and are components of key regulatory complexes, such as transcriptional regulatory and chromatin remodeling complexes. Under hypoxia, these proteins are synthesized and retained in the cytosol. Upon reoxygenation, they relocalize effectively to their normal cellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cell periphery. The resumption of the normal cellular locations of many proteins can occur even when protein synthesis is inhibited. Furthermore, we show that the changes in protein distribution induced by hypoxia follow a slower trajectory than those induced by reoxygenation. These results show that the regulation of protein localization is a common and potentially dominant mechanism underlying oxygen signaling and regulation. These results may have broad implications in understanding oxygen signaling and hypoxia responses in higher eukaryotes such as humans.

scholarly journals A small molecule-directed approach to control protein localization and function

Yeast ◽  
2008 ◽  
Vol 25 (8) ◽  
pp. 577-594 ◽  
Author(s):  
Prasanthi Geda ◽  
Srikanth Patury ◽  
Jun Ma ◽  
Nike Bharucha ◽  
Craig J. Dobry ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Localization ◽  
And Function ◽  
Control Protein

scholarly journals Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis

2021 ◽  
Author(s):  
Corelle A. Z. Rokicki ◽  
James R. Brenner ◽  
Alexander H. Dills ◽  
Julius J. Judd ◽  
Jemila C. Kester ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Protein Localization ◽  
Subcellular Fractionation ◽  
Polar Regions ◽  
Intracellular Membrane ◽  
Membrane Domain ◽  
Associated Proteins ◽  
Proteomic Analyses

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under non-growing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized biosynthesis of envelope components. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by independent experimental methods. Furthermore, some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains, each producing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed polar fluorescence patterns characteristic of IMD proteins. Twenty of these had previously been suggested to localize to the IMD based on proteomic data. Of the nine remaining IMD candidate proteins, three were confirmed by biochemical methods to be associated with the IMD. Taken together, this new co-localization strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins. Importance The intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis. However, proteomics on its own is unlikely to detect every IMD-associated protein because of technical and biological limitations. Here, we describe fluorescent protein co-localization as an alternative, independent approach. Using a combination of fluorescence microscopy, proteomics, and subcellular fractionation, we identified three new proteins associated with the IMD. Such a robust method to rigorously define IMD proteins will benefit future investigations to decipher the synthesis, maintenance and functions of this membrane domain, and help delineate a more general mechanisms of subcellular protein localization in mycobacteria.

scholarly journals Microtubule-associated Proteins Regulate Microtubule Function as the Track for Intracellular Membrane Organelle Transports.

1996 ◽  
Vol 21 (5) ◽  
pp. 283-295 ◽  
Author(s):  
Reiko Sato-Harada ◽  
Shigeo Okabe ◽  
Takashige Umeyama ◽  
Yoshimitsu Kanai ◽  
Nobutaka Hirokawa
Keyword(s):  
Intracellular Membrane ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Membrane Organelle ◽  
Microtubule Function

scholarly journals Phosphoinositides in Retinal Function and Disease

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 866 ◽  
Author(s):  
Theodore G. Wensel
Keyword(s):  
Membrane Trafficking ◽  
New Technologies ◽  
Second Messengers ◽  
Regulatory Pathways ◽  
Peripheral Membrane Proteins ◽  
New Information ◽  
Mammalian Retina ◽  
Pigmented Epithelium ◽  
Associated Proteins ◽  
And Control

Phosphatidylinositol and its phosphorylated derivatives, the phosphoinositides, play many important roles in all eukaryotic cells. These include modulation of physical properties of membranes, activation or inhibition of membrane-associated proteins, recruitment of peripheral membrane proteins that act as effectors, and control of membrane trafficking. They also serve as precursors for important second messengers, inositol (1,4,5) trisphosphate and diacylglycerol. Animal models and human diseases involving defects in phosphoinositide regulatory pathways have revealed their importance for function in the mammalian retina and retinal pigmented epithelium. New technologies for localizing, measuring and genetically manipulating them are revealing new information about their importance for the function and health of the vertebrate retina.

Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap

2000 ◽  
Vol 113 (22) ◽  
pp. 3907-3919 ◽  
Author(s):  
A.S. Infante ◽  
M.S. Stein ◽  
Y. Zhai ◽  
G.G. Borisy ◽  
G.G. Gundersen
Keyword(s):  
Focal Adhesions ◽  
Cell Types ◽  
Cell Models ◽  
Microtubule Stability ◽  
Associated Proteins ◽  
Atp Breakdown ◽  
The Stability ◽  
Conventional Kinesin

Many cell types contain a subset of long-lived, ‘stable’ microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca(2+)-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150(Glued) and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.

scholarly journals An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

2016 ◽  
Vol 27 (3) ◽  
pp. 549-571 ◽  
Author(s):  
Jun Liu ◽  
Yudou He ◽  
Imaan Benmerzouga ◽  
William J. Sullivan ◽  
Naomi S. Morrissette ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Mitotic Spindle ◽  
Functional Differentiation ◽  
Cortical Microtubules ◽  
Human Parasite ◽  
Cellular Morphogenesis ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
The Stability ◽  
Directional Transport

Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy.

scholarly journals Global, quantitative and dynamic mapping of protein subcellular localization

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Daniel N Itzhak ◽  
Stefka Tyanova ◽  
Jürgen Cox ◽  
Georg HH Borner
Keyword(s):  
Subcellular Localization ◽  
Protein Function ◽  
Cell Biology ◽  
Dynamic Capabilities ◽  
Protein Translocation ◽  
Protein Localization ◽  
Quantitative Model ◽  
Dynamic Mapping ◽  
Global Mapping ◽  
Control Protein

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

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