Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins

Joglekar, Ajit P.; Salmon, Edward D.; Bloom, Kerry

doi:10.1016/s0091-679x(08)85007-8

Cited by 56 publications

(81 citation statements)

References 55 publications

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“…In histograms of fluorescence intensities, we observed a trail in the distribution toward higher fluorescence intensities that might indicate formation of complexes with more subunits (see Supplemental Figure 1, A-F). To confirm these conclusion, we also GFP-tagged kinetochore proteins Cse4 and Spc105 as markers to quantify in vivo protein numbers (Joglekar et al, 2008). One kinetochore cluster of Cse4-GFP represents the fluorescence of 32 GFP molecules, whereas a kinetochore cluster of Spc105-GFP represents 80 GFP molecules.…”

Section: All Torc2 Components Localize As Oligomeric Foci To the Plasmentioning

confidence: 86%

TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain

Berchtold

Walther

2009

MBoC

181

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The conserved target of rapamycin (TOR) kinases regulate many aspects of cellular physiology. They exist in two distinct complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2), that posses both overlapping and distinct components. TORC1 and TORC2 respond differently to the drug rapamycin and have different cellular functions: whereas the rapamycin-sensitive TORC1 controls many aspects of cell growth and has been characterized in great detail, the TOR complex 2 is less understood and regulates actin polymerization, cell polarity, and ceramide metabolism. How signaling specificity and discrimination between different input signals for the two kinase complexes is achieved is not understood. Here, we show that TORC1 and TORC2 have different localizations in Saccharomyces cerevisiae. TORC1 is localized exclusively to the vacuolar membrane, whereas TORC2 is localized dynamically in a previously unrecognized plasma membrane domain, which we term membrane compartment containing TORC2 (MCT). We find that plasma membrane localization of TORC2 is essential for viability and mediated by lipid binding of the C-terminal domain of the Avo1 subunit. From these data, we suggest that the TOR complexes are spatially separated to determine downstream signaling specificity and their responsiveness to different inputs. INTRODUCTIONDuring cell growth and division, many physiological processes need to be coordinated and modified according to nutrient availability. Signaling through target of rapamycin (TOR) kinases plays a central role in this regulation. Even though TOR kinases are related to phosphoinositide lipid kinases, they are Ser/Thr protein kinases, with a small number of known targets that regulate many cellular processes. Together, the diverse TOR signaling outputs regulate cell growth, both spatially and temporally (De Virgilio and Loewith, 2006;Reiling and Sabatini, 2006;Wullschleger et al., 2006).The characterization of TOR signaling was greatly aided by the discovery of the antifungal and immunosuppressant macrocyclic lactone rapamycin. The search for targets of this drug in the yeast Saccharomyces cerevisiae lead to the discovery of the TOR kinases and subsequently helped to identify their molecular function (Heitman et al., 1991).The rapamycin-sensitive branch of TOR signaling regulates processes that collectively modulate the rate of cell growth. TOR signaling is active under conditions of excess nutrients, blocking catabolic processes, such as autophagy and stress responses. In contrast, exposure to rapamycin or withdrawal of nitrogen or carbon sources lead to inactivation of TOR signaling and down-regulation of anabolic processes, such as protein synthesis. Together these processes coordinate cell growth with nutrient availability (De Virgilio and Loewith, 2006;Wullschleger et al., 2006). This function of TOR signaling is performed by TOR complex 1 (TORC1) that consists of the kinase plus three additional subunits in yeast, named Lst8, Kog1, and Tco89 or two subunits named raptor and mLst8 in mammals. Its...

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Section: All Torc2 Components Localize As Oligomeric Foci To the Plasmentioning

confidence: 86%

TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain

Berchtold

Walther

2009

MBoC

181

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“…However, possible interference of the fluorescent tag with protein function remains a concern. In addition, quantitative measurements of fluorescence signals are complicated by photo‐bleaching, quenching of signal intensity, and the difficulty of dependable standardization (Joglekar et al , 2008; Lawrimore et al , 2011; Coffman & Wu, 2012). Thus, we are reassured by the good concordance between results obtained through use of the two complementary approaches.…”

Section: Discussionmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

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“…Structural analysis in vitro has shown that a Dam1 ring contains at least 16 copies of the complex (5,6). To test whether SpDam1 forms rings around MTs, we measured the copy number of Dam1 in individual spots on cytoplasmic MTs, using an established quantitative fluorescence microscopy approach (18,25,27). We examined strains expressing tri or di-GFP-tagged Dam1 complexes, and deduced the copy number of Dam1 ( Fig S2 and Materials and Methods).…”

Section: Identification Of Discrete Dam1 Assemblies On Cytoplasmic Mtmentioning

confidence: 99%

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

Gao

Courthéoux

Gachet

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The Dam1 complex is a kinetochore component that couples chromosomes to the dynamic ends of kinetochore microtubules (kMTs). Work in the budding yeast Saccharomyces cerevisiae has shown that the Dam1 complex forms a 16-unit ring encircling and tracking the tip of a MT in vitro, consistent with its cellular function as a coupler. Dam1 also forms smaller, nonring patches in vitro that track the dynamic ends of MTs. However, the identity of Dam1’s functional form in vivo remains unknown. Here we report a comprehensive in vivo characterization of Dam1 in the fission yeast Schizosaccharomyces pombe . In addition to their dense localizations on kinetochores and spindle MTs during mitosis, we identify that Dam1 is also localized onto cytoplasmic MTs as discrete spots in interphase, providing the unique opportunity to analyze Dam1 oligomers at the single-particle resolution in live cells. Such analysis shows that each oligomer contains one to five copies of Dam1, and is able to “switch-rail” while moving along MTs, precluding the possibility of a 16-unit encircling structure. Dam1 patches track the plus ends of the shortening, but not the elongating, MTs and retard MT depolymerization. Together with Mal3, the EB1-like MT-interacting protein, cytoplasmic Dam1 plays an important role in maintaining proper cell shape. In mitosis, kinetochore-associated Dam1 appears to facilitate kMT depolymerization. Together, our findings suggest that patches, instead of rings, are the physiologically functional forms of Dam1 in pombe. Our findings help establish the benchmark parameters of the Dam1 coupler and elucidate the mechanism of its functions.

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Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins

Cited by 56 publications

References 55 publications

TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain

TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

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