scholarly journals Both Clathrin-Mediated and Membrane Microdomain-Associated Endocytosis Contribute to the Cellular Adaptation to Hyperosmotic Stress in Arabidopsis

2021 ◽  
Vol 22 (22) ◽  
pp. 12534
Author(s):  
Zheng Wu ◽  
Chengyu Fan ◽  
Yi Man ◽  
Yue Zhang ◽  
Ruili Li ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Fluorescent Protein ◽  
Abiotic Factors ◽  
Central Compartment ◽  
Hyperosmotic Stress ◽  
Animal Cells ◽  
Cellular Adaptation ◽  
Membrane Microdomain ◽  
Hydrophobic Barrier ◽  
Green Fluorescent

As sessile organisms, plants must directly deal with an often complex and adverse environment in which hyperosmotic stress is one of the most serious abiotic factors, challenging cellular physiology and integrity. The plasma membrane (PM) is the hydrophobic barrier between the inside and outside environments of cells and is considered a central compartment in cellular adaptation to diverse stress conditions through dynamic PM remodeling. Endocytosis is a powerful method for rapid remodeling of the PM. In animal cells, different endocytic pathways are activated in response to osmotic stress, while only a few reports are related to the endocytosis response pathway and involve a mechanism in plant cells upon hyperosmotic stress. In this study, using different endocytosis inhibitors, the microdomain-specific dye di-4-ANEPPDHQ, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), and confocal microscopy, we discovered that internalized Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) increased under hyperosmotic conditions, accompanied by decreased fluorescence intensity of CLC-GFP at the PM. CLC-GFP tended to have higher diffusion coefficients and a fraction of CLC-GFP molecules underwent slower diffusion upon hyperosmotic stress. Meanwhile, an increased motion range of CLC-GFP was found under hyperosmotic treatment compared with the control. In addition, the order of the PM decreased, but the order of the endosome increased when cells were in hyperosmotic conditions. Hence, our results demonstrated that clathrin-mediated endocytosis and membrane microdomain-associated endocytosis both participate in the adaptation to hyperosmotic stress. These findings will help to further understand the role and the regulatory mechanism involved in plant endocytosis in helping plants adapt to osmotic stress.

scholarly journals Septin Function inCandida albicansMorphogenesis

2002 ◽  
Vol 13 (8) ◽  
pp. 2732-2746 ◽  
Author(s):  
Amy J. Warenda ◽  
James B. Konopka
Keyword(s):  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Wild Type ◽  
Animal Cells ◽  
Hyphal Morphogenesis ◽  
Hyphal Formation ◽  
Green Fluorescent ◽  
Mutant Phenotypes ◽  
Germ Tubes ◽  
Selection Of

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 andCDC12 septins are essential for viability. In contrast, the C. albicans cdc10Δ and cdc11Δ mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. Thecdc11Δ mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.

scholarly journals Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex

1997 ◽  
Vol 138 (3) ◽  
pp. 629-641 ◽  
Author(s):  
Janet L. Carminati ◽  
Tim Stearns
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Dynamic Properties ◽  
Yeast Cells ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Animal Cells ◽  
Green Fluorescent ◽  
Mutant Cells

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)–tubulin fusion protein to observe microtubules in living yeast cells. GFP–tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

scholarly journals The Yeast Inositol Polyphosphate 5-Phosphatases Inp52p and Inp53p Translocate to Actin Patches following Hyperosmotic Stress: Mechanism for Regulating Phosphatidylinositol 4,5-Bisphosphate at Plasma Membrane Invaginations

2000 ◽  
Vol 20 (24) ◽  
pp. 9376-9390 ◽  
Author(s):  
Lisa M. Ooms ◽  
Brad K. McColl ◽  
Fenny Wiradjaja ◽  
A. P. W. Wijayaratnam ◽  
Paul Gleeson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Hyperosmotic Stress ◽  
Resting Cells ◽  
Inositol Polyphosphate ◽  
Rhodamine Phalloidin ◽  
Phosphatase Domain ◽  
Rich Domain ◽  
Green Fluorescent

ABSTRACT The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and PtdIns(3,5)P2. Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P2 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P2 and PtdIns(4,5)P2 at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.

scholarly journals In yeast, loss of Hog1 leads to osmosensitivity of autophagy

2006 ◽  
Vol 394 (1) ◽  
pp. 153-161 ◽  
Author(s):  
Tanja Prick ◽  
Michael Thumm ◽  
Karl Köhrer ◽  
Dieter Häussinger ◽  
Stephan Vom Dahl
Keyword(s):  
Osmotic Stress ◽  
Fluorescent Protein ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
Cell Swelling ◽  
Nitrogen Deprivation ◽  
Integrin Receptors ◽  
Mitogen Activated Protein ◽  
Mammalian Liver ◽  
Green Fluorescent

In mammalian liver, proteolysis is regulated by the cellular hydration state in a microtubule- and p38MAPK (p38 mitogen-activated protein kinase)-dependent fashion. Osmosensing in liver cells towards proteolysis is achieved by activation of integrin receptors. The yeast orthologue of p38MAPK is Hog1 (high-osmolarity glycerol 1), which is involved in the hyperosmotic-response pathway. Since it is not known whether starvation-induced autophagy in yeast is osmosensitive and whether Hog1 is involved in this process, we performed fluorescence microscopy experiments. The hog1Δ cells exhibited a visible decrease of autophagy in hypo-osmotic and hyperosmotic nitrogen-starvation medium as compared with normo-osmolarity, as determined by GFP (green fluorescent protein)–Atg8 (autophagy-related 8) fluorescence. Western blot analysis of GFP–Atg8 degradation showed that WT (wild-type) cells maintained a stable autophagic activity over a broad osmolarity range, whereas hog1Δ cells showed an impaired autophagic actitivity during hypo- and hyper-osmotic stress. In [3H]leucine-pre-labelled yeast cells, the proteolysis rate was osmodependent only in hog1Δ cells. Neither maturation of pro-aminopeptidase I nor vitality was affected by osmotic stress in either yeast strain. In contrast, rapamycin-dependent autophagy, as measured by degradation of GFP–Atg8, did not significantly respond to hypo-osmotic or hyperosmotic stress in hog1Δ or WT cells. We conclude that Hog1 plays a role in the stabilization machinery of nitrogen-deprivation-induced autophagy in yeast cells during ambient osmolarity changes. This could be an analogy to the p38MAPK pathway in mammalian liver, where osmosensing towards p38MAPK is required for autophagy regulation by hypo-osmotic or amino-acid-induced cell swelling. A phenotypic difference is observed in rapamycin-induced autophagy, which does not seem to respond to extracellular osmolarity changes in hog1Δ cells.

scholarly journals An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Sarah Borg ◽  
Felix Popp ◽  
Julia Hofmann ◽  
Heinrich Leonhardt ◽  
Ulrich Rothbauer ◽  
...  
Keyword(s):  
Protein Function ◽  
Fluorescent Protein ◽  
Cellular Structures ◽  
Animal Cells ◽  
Living Plant ◽  
Content Type ◽  
Live Bacteria ◽  
Green Fluorescent ◽  
Intracellular Targets ◽  
Intracellular Compartmentalization

ABSTRACT Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular “nanotraps,” which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacteriumMagnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW1-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria.IMPORTANCE   Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular “nanotraps” in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks.

scholarly journals Aspergillus glaucus Aquaglyceroporin GeneglpFConfers High Osmosis Tolerance in Heterologous Organisms

2015 ◽  
Vol 81 (19) ◽  
pp. 6926-6937 ◽  
Author(s):  
Xiao-Dan Liu ◽  
Yi Wei ◽  
Xiao-Yang Zhou ◽  
Xue Pei ◽  
Shi-Hong Zhang
Keyword(s):  
Osmotic Stress ◽  
Fluorescent Protein ◽  
Salt Resistance ◽  
Fungal Species ◽  
Content Type ◽  
Aspergillus Glaucus ◽  
Water Glycerol ◽  
Complementary Strain ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

ABSTRACTAquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, aglpFgene from the halophilic fungusAspergillus glaucus(AgglpF) was verified to be a channel of water or glycerol inXenopus laevisoocytes and was further functionally analyzed in three heterologous systems. InSaccharomyces cerevisiae, cells overexpressingAgglpFpossessed significant tolerance of drought, salt, and certain metal ions.AgglpFwas then characterized in the filamentous fungus ofNeurospora crassa. Based on theN. crassaaquaporin gene (NcAQP) disruption mutant (the Δaqpmutant), a series of complementary strains carryingNcAQPandAgglpFand three asparagine-proline-alanine-gene (NPA)-deletedAgglpFfragments were created. As revealed by salt resistance analysis, theAgglpFcomplementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in theAgglpFcomplementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting thatAgglpFfunctions in plants. Indeed, whenAgglpFwas expressed inArabidopsis thaliana, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance.

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

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

<p>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 <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> 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.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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