Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint

The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD–ZW10–Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1–noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein–hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest.

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New insight into bulb dynamics in the vacuolar lumen of Arabidopsis cells

Botany ◽

10.1139/cjb-2018-0009 ◽

2018 ◽

Vol 96 (8) ◽

pp. 511-520 ◽

Cited By ~ 2

Author(s):

Mst Hur Madina ◽

Huanquan Zheng ◽

Hugo Germain

Keyword(s):

Fluorescence Recovery After Photobleaching ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Effector Protein ◽

Protein Distribution ◽

Marker Protein ◽

Plant Vacuoles ◽

Green Fluorescent ◽

Insight Into ◽

Arabidopsis Cells

Plant vacuoles are multifunctional organelles with dynamic and transient membranous structures, such as trans-vacuolar strands and bulbs. Bulbs are highly mobile structures that travel along trans-vacuolar strands. A candidate effector protein from Melampsora larici-populina (Mlp124357) fused with the enhanced green fluorescent protein (eGFP) was used to investigate the properties of central vacuoles and their bulbs. We discovered the coexistence of two bulb populations in Arabidopsis cells. In addition to previously-described bulbs, which present even marker protein distribution on the bulb surface, we discerned bulbs displaying irregular fusion protein distribution. Using fluorescence recovery after photobleaching (FRAP), we also demonstrated that bulbs do not exchange proteins with the tonoplast once they are formed. These results show that more than one type of bulb may co-exist in the same cell and provide evidence of micro-domains on the bulb surface. They also reveal that proteins do not flow freely from the tonoplast to the bulb membrane, giving new insight into the biology of tonoplast-derived substructures.

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Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

Optics Express ◽

10.1364/oe.14.009815 ◽

2006 ◽

Vol 14 (21) ◽

pp. 9815 ◽

Cited By ~ 5

Author(s):

Alberto Diaspro ◽

Silke Krol ◽

Barbara Campanini ◽

Fabio Cannone ◽

Giuseppe Chirico

Keyword(s):

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Gfp Fluorescence ◽

Green Fluorescent

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Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽

10.3390/v13040632 ◽

2021 ◽

Vol 13 (4) ◽

pp. 632

Author(s):

Yingyun Cai ◽

Shuiqing Yu ◽

Ying Fang ◽

Laura Bollinger ◽

Yanhua Li ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Lines ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Infectious Clone ◽

Hemorrhagic Fever ◽

Simian Hemorrhagic Fever Virus ◽

Hemorrhagic Fever Virus ◽

Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

Molecular and Cellular Biology ◽

10.1128/mcb.25.12.4977-4992.2005 ◽

2005 ◽

Vol 25 (12) ◽

pp. 4977-4992 ◽

Cited By ~ 100

Author(s):

Hao G. Nguyen ◽

Dharmaraj Chinnappan ◽

Takeshi Urano ◽

Katya Ravid

Keyword(s):

Chromosome Segregation ◽

Fluorescent Protein ◽

Point Mutations ◽

Degradation Pathway ◽

Aurora B ◽

Stable Form ◽

Wild Type ◽

Green Fluorescent ◽

Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

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Nuclear and subnuclear targeting sequences of the protein phosphatase-1 regulator NIPP1

Journal of Cell Science ◽

10.1242/jcs.113.21.3761 ◽

2000 ◽

Vol 113 (21) ◽

pp. 3761-3768 ◽

Cited By ~ 5

Author(s):

I. Jagiello ◽

A. Van Eynde ◽

V. Vulsteke ◽

M. Beullens ◽

A. Boudrez ◽

...

Keyword(s):

Active Transport ◽

Protein Phosphatase ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Protein Phosphatase 1 ◽

Splicing Factors ◽

Nuclear Speckles ◽

Nuclear Retention ◽

Nuclear Localization Signals ◽

Green Fluorescent

NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the ‘ForkHead-Associated’ domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.

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