scholarly journals Analysis of interphase node proteins in fission yeast by quantitative and super resolution fluorescence microscopy

2017 ◽  
Author(s):  
Matthew Akamatsu ◽  
Yu Lin ◽  
Joerg Bewersdorf ◽  
Thomas D. Pollard
Keyword(s):  
Cell Cycle ◽  
Confocal Microscopy ◽  
Fission Yeast ◽  
Super Resolution ◽  
Steady Increase ◽  
Contractile Ring ◽  
Two Phases ◽  
Super Resolution Microscopy

AbstractWe used quantitative confocal microscopy and FPALM super resolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes, multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases, a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase.Highlight summaryFPALM super resolution microscopy and quantitative confocal microscopy reveal that interphase nodes, the precursors to the fission yeast cytokinetic contractile ring, are discrete unitary structures with defined sizes and ratios of component proteins. Type 1 nodes disassemble during mitosis, but type 2 nodes remain intact throughout the cell cycle.

scholarly journals Analysis of interphase node proteins in fission yeast by quantitative and superresolution fluorescence microscopy

2017 ◽  
Vol 28 (23) ◽  
pp. 3203-3214 ◽  
Author(s):  
Matthew Akamatsu ◽  
Yu Lin ◽  
Joerg Bewersdorf ◽  
Thomas D. Pollard
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Steady Increase ◽  
Contractile Ring ◽  
Superresolution Microscopy ◽  
Quantitative Measurements ◽  
Two Phases ◽  
Rho Gef

We used quantitative confocal microscopy and FPALM superresolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes—multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle, seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis, the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases—a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase.

scholarly journals Cytokinetic nodes in fission yeast arise from two distinct types of nodes that merge during interphase

2014 ◽  
Vol 204 (6) ◽  
pp. 977-988 ◽  
Author(s):  
Matthew Akamatsu ◽  
Julien Berro ◽  
Kai-Ming Pu ◽  
Irene R. Tebbs ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Fusion Proteins ◽  
Contractile Ring ◽  
Guanosine Triphosphate ◽  
Quantitative Measurements ◽  
Capture Mechanism ◽  
Septation Initiation Network ◽  
Stationary Type

We investigated the assembly of cortical nodes that generate the cytokinetic contractile ring in fission yeast. Observations of cells expressing fluorescent fusion proteins revealed two types of interphase nodes. Type 1 nodes containing kinase Cdr1p, kinase Cdr2p, and anillin Mid1p form in the cortex around the nucleus early in G2. Type 2 nodes with protein Blt1p, guanosine triphosphate exchange factor Gef2p, and kinesin Klp8p emerge from contractile ring remnants. Quantitative measurements and computer simulations showed that these two types of nodes come together by a diffuse-and-capture mechanism: type 2 nodes diffuse to the equator and are captured by stationary type 1 nodes. During mitosis, cytokinetic nodes with Mid1p and all of the type 2 node markers incorporate into the contractile ring, whereas type 1 nodes with Cdr1p and Cdr2p follow the separating nuclei before dispersing into the cytoplasm, dependent on septation initiation network signaling. The two types of interphase nodes follow parallel branches of the pathway to prepare nodes for cytokinesis.

scholarly journals Myosins generate contractile force and maintain organization in the cytokinetic contractile ring

2021 ◽  
Author(s):  
Zachary A. McDargh ◽  
Shuyuan Wang ◽  
Harvey F. Chin ◽  
Sathish Thiyagarajan ◽  
Erdem Karatekin ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Striated Muscle ◽  
Protein Complexes ◽  
Myosin Ii ◽  
Force Production ◽  
Super Resolution ◽  
Building Blocks ◽  
Contractile Ring ◽  
Ring Model ◽  
Self Assembling

During cytokinesis, cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here, we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.

scholarly journals Effect of Nano-Sized Cavities in SAPO-34 Zeolite on Thermodynamics of Adsorbed Gas Mixtures

Nanomaterials ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 672 ◽  
Author(s):  
Fei Wang ◽  
Yasukazu Kobayashi ◽  
Yuxin Li ◽  
Dezheng Wang ◽  
Yao Wang
Keyword(s):  
Langmuir Isotherm ◽  
Acid Sites ◽  
Adsorbed Gas ◽  
Ideal Adsorbed Solution Theory ◽  
Binary Gas Mixture ◽  
Langmuir Isotherm Model ◽  
Adsorbed Solution ◽  
Two Phases

Adsorption of dimethyl ether and ethene in SAPO-34 zeolite with the calorimetric (adsorption heat versus coverage) curve measured together with the adsorption isotherm showed two phases of adsorption: first, Type 1 adsorption on acid sites, and second, Type 2 adsorption elsewhere in the cages by physisorption that continued with increasing pressure. Binary gas mixture experiments showed that only the ideal adsorbed solution theory (IAST) gave correct surface concentrations, while the multicomponent Langmuir isotherm for competitive adsorption was incorrect even though the acid site concentration was the same for the adsorbates. This is because the adsorption occurred in two adsorption phases while the Langmuir isotherm model is based on a single adsorption phase.

scholarly journals Diagnosis of Neuropathy and Risk Factors for Corneal Nerve Loss in Type 1 and Type 2 Diabetes: A Corneal Confocal Microscopy Study

Diabetes Care ◽  
2020 ◽  
Vol 44 (1) ◽  
pp. 150-156
Author(s):  
Maryam Ferdousi ◽  
Alise Kalteniece ◽  
Shazli Azmi ◽  
Ioannis N. Petropoulos ◽  
Georgios Ponirakis ◽  
...  
Keyword(s):  
Risk Factors ◽  
Type 2 Diabetes ◽  
Confocal Microscopy ◽  
Microscopy Study ◽  
Corneal Confocal Microscopy ◽  
Corneal Nerve

scholarly journals Drug Testing for Residual Progression of Diabetic Kidney Disease in Mice Beyond Therapy with Metformin, Ramipril, and Empagliflozin

2020 ◽  
Vol 31 (8) ◽  
pp. 1729-1745 ◽  
Author(s):  
Manga Motrapu ◽  
Monika Katarzyna Świderska ◽  
Irene Mesas ◽  
Julian Aurelio Marschner ◽  
Yutian Lei ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Drug Testing ◽  
Glucose Transporter ◽  
Super Resolution ◽  
Renin Angiotensin System ◽  
Protective Effects ◽  
Angiotensin System ◽  
Glucose Transporter 2 ◽  
Super Resolution Microscopy

BackgroundProgression of CKD in type 2 diabetes, despite dual inhibition of sodium-glucose transporter-2 and the renin-angiotensin system, remains a concern. Bromoindirubin-3′-oxime (BIO), previously reported to promote podocyte survival and regeneration, is a candidate additional drug to elicit renoprotective effects beyond therapy with metformin, ramipril, and empagliflozin (MRE). Evaluating a drug with standard therapeutics more closely mimics the clinical setting than evaluating the drug alone.MethodsUninephrectomized BKS-Lepr−/− (db/db) mice treated with or without MRE served as a model of progressive CKD in type 2 diabetes. Mice on or off MRE were randomized to only 4 weeks of add-on BIO or vehicle. The primary end point was slope of GFR (ΔGFR).ResultsFour weeks of MRE treatment alone did not affect ΔGFR, but significantly attenuated hyperglycemia, albuminuria, and glomerulosclerosis and increased podocyte filtration slit density, as assessed by STED super-resolution microscopy upon tissue clearing. BIO alone improved albuminuria, podocyte density in superficial and juxtamedullary nephrons, and podocyte filtration slit density. MRE+BIO combination therapy had additive protective effects on ΔGFR, glomerulosclerosis, podocyte density in juxtamedullary nephrons, and filtration slit density.ConclusionsAdd-on treatment with BIO for only 4 weeks attenuates progression of CKD beyond MRE therapy in mice with type 2 diabetes. Additional drug combinations may help to further delay ESKD in type 2 diabetes.

scholarly journals The Clp1/Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1

2008 ◽  
Vol 181 (1) ◽  
pp. 79-88 ◽  
Author(s):  
Dawn M. Clifford ◽  
Benjamin A. Wolfe ◽  
Rachel H. Roberts-Galbraith ◽  
W. Hayes McDonald ◽  
John R. Yates ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Cycle Control ◽  
Physical Property ◽  
Cyclin Dependent Kinase ◽  
Contractile Ring ◽  
Protein Mobility ◽  
Functional Consequences

Cdc14 phosphatases antagonize cyclin-dependent kinase–directed phosphorylation events and are involved in several facets of cell cycle control. We investigate the role of the fission yeast Cdc14 homologue Clp1/Flp1 in cytokinesis. We find that Clp1/Flp1 is tethered at the contractile ring (CR) through its association with anillin-related Mid1. Fluorescent recovery after photobleaching analyses indicate that Mid1, unlike other tested CR components, is anchored at the cell midzone, and this physical property is likely to account for its scaffolding role. By generating a mutation in mid1 that selectively disrupts Clp1/Flp1 tethering, we reveal the specific functional consequences of Clp1/Flp1 activity at the CR, including dephosphorylation of the essential CR component Cdc15, reductions in CR protein mobility, and CR resistance to mild perturbation. Our evidence indicates that Clp1/Flp1 must interact with the Mid1 scaffold to ensure the fidelity of Schizosaccharomyces pombe cytokinesis.

scholarly journals Human Immunodeficiency Virus Type 1 Vpr Induces Cell Cycle G2 Arrest through Srk1/MK2-Mediated Phosphorylation of Cdc25

2007 ◽  
Vol 82 (6) ◽  
pp. 2904-2917 ◽  
Author(s):  
Sylvain Huard ◽  
Robert T. Elder ◽  
Dong Liang ◽  
Ge Li ◽  
Richard Y. Zhao
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
Fission Yeast ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Elongation ◽  
Mammalian Cells ◽  
Nuclear Exclusion ◽  
Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G2 arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G2 arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G2/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G2 arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G2 delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G2/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G2 arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G2 arrest at least in part through a Srk1/MK2-mediated mechanism.

scholarly journals Reticulon and CLIMP-63 control nanodomain organization of peripheral ER tubules

2019 ◽  
Author(s):  
Guang Gao ◽  
Chengjia Zhu ◽  
Emma Liu ◽  
Ivan R. Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
High Speed ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Preferential Segregation ◽  
Stimulated Emission Depletion ◽  
Super Resolution Microscopy ◽  
Local Accumulation

AbstractThe endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon and CLIMP-63, respectively. Here, STimulated Emission Depletion (STED) super-resolution microscopy shows that reticulon and CLIMP-63 also control the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ERmoxGFP, membrane Sec61βGFP, knock-in calreticulin-GFP and antibody-labeled ER resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. Reticulon segregates away from and restricts lumenal blob length while CLIMP-63 associates with and increases lumenal blob length. Reticulon and CLIMP-63 also regulate the nanodomain distribution of ER resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that reticulon and CLIMP-63 control nanoscale compartmentalization of lumenal flow in peripheral ER tubules. Reticulon enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER shaping proteins reticulon and CLIMP-63 therefore control lumenal ER nanodomain dynamics, heterogeneity and interaction with ER resident proteins in peripheral ER tubules.

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