scholarly journals Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

1998 ◽  
Vol 140 (2) ◽  
pp. 355-366 ◽  
Author(s):  
John Lippincott ◽  
Rong Li
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Sequence Similarity ◽  
Myosin Ii ◽  
Ring Structure ◽  
Binding Activity ◽  
Filamentous Actin ◽  
Green Fluorescent

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.

scholarly journals Dual Function of Cyk2, a cdc15/PSTPIP Family Protein, in Regulating Actomyosin Ring Dynamics and Septin Distribution

1998 ◽  
Vol 143 (7) ◽  
pp. 1947-1960 ◽  
Author(s):  
John Lippincott ◽  
Rong Li
Keyword(s):  
Fluorescent Protein ◽  
Budding Yeast ◽  
Sequence Similarity ◽  
Dual Function ◽  
Size Change ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Ring ◽  
Green Fluorescent ◽  
And Function

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.

scholarly journals Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8191-8200 ◽  
Author(s):  
Philippe Bastin ◽  
Thomas H. MacRae ◽  
Susan B. Francis ◽  
Keith R. Matthews ◽  
Keith Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Reporter ◽  
Mutant Proteins ◽  
Green Fluorescent ◽  
A Minor ◽  
Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

scholarly journals Daip1, a Dictyostelium Homologue of the Yeast Actin-Interacting Protein 1, Is Involved in Endocytosis, Cytokinesis, and Motility

1999 ◽  
Vol 146 (2) ◽  
pp. 453-464 ◽  
Author(s):  
Angelika Konzok ◽  
Igor Weber ◽  
Evelyn Simmeth ◽  
Ulrike Hacker ◽  
Markus Maniak ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Gene Replacement ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Phagocytic Cups ◽  
Green Fluorescent

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.

scholarly journals The Sudden Recruitment of γ-Tubulin to the Centrosome at the Onset of Mitosis and Its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules

1999 ◽  
Vol 146 (3) ◽  
pp. 585-596 ◽  
Author(s):  
Alexey Khodjakov ◽  
Conly L. Rieder
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Green Fluorescent ◽  
Dynamic Exchange ◽  
Two Populations

γ-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a γ-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of γ-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of γ-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of γ-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated γ-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated γ-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional γ-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of γ-tubulin than during interphase.

Hypoxia induces major effects on cell cycle kinetics and protein expression in Drosophila melanogaster embryos

2005 ◽  
Vol 288 (2) ◽  
pp. R511-R521 ◽  
Author(s):  
R. M. Douglas ◽  
R. Farahani ◽  
P. Morcillo ◽  
A. Kanaan ◽  
T. Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Cycle Length ◽  
Fluorescent Protein ◽  
Fruit Fly ◽  
Cyclin A ◽  
S Phase ◽  
Green Fluorescent ◽  
Cell Cycle Length

Hypoxia induces a stereotypic response in Drosophila melanogaster embryos: depending on the time of hypoxia, embryos arrest cell cycle activity either at metaphase or just before S phase. To understand the mechanisms underlying hypoxia-induced arrest, two kinds of experiments were conducted. First, embryos carrying a kinesin-green fluorescent protein construct, which permits in vivo confocal microscopic visualization of the cell cycle, showed a dose-response relation between O2 level and cell cycle length. For example, mild hypoxia (Po2 ∼55 Torr) had no apparent effect on cell cycle length, whereas severe hypoxia (Po2 ∼25–35 Torr) or anoxia (Po2 = 0 Torr) arrested the cell cycle. Second, we utilized Drosophila embryos carrying a heat shock promoter driving the string ( cdc25) gene (HS-STG3), which permits synchronization of embryos before the start of mitosis. Under conditions of anoxia, we induced a stabilization or an increase in the expression of several G1/S (e.g., dE2F1, RBF2) and G2/M (e.g., cyclin A, cyclin B, dWee1) proteins. This study suggests that, in fruit fly embryos, 1) there is a dose-dependent relationship between cell cycle length and O2 levels in fruit fly embryos, and 2) stabilized cyclin A and E2F1 are likely to be the mediators of hypoxia-induced arrest at metaphase and pre-S phase.

scholarly journals Microtubule Organization Requires Cell Cycle-dependent Nucleation at Dispersed Cytoplasmic Sites: Polar and Perinuclear Microtubule Organizing Centers in the Plant Pathogen Ustilago maydis

2003 ◽  
Vol 14 (2) ◽  
pp. 642-657 ◽  
Author(s):  
Anne Straube ◽  
Marianne Brill ◽  
Berl R. Oakley ◽  
Tetsuya Horio ◽  
Gero Steinberg
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Organization ◽  
Microtubule Organizing Centers ◽  
Nucleation Sites ◽  
Cycle Dependent ◽  
Green Fluorescent

Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungusUstilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding γ-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the γ-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited γ-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

2008 ◽  
Vol 410 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Fui-Ching Tan ◽  
Qi Cheng ◽  
Kaushik Saha ◽  
Ilka U. Heinemann ◽  
Martina Jahn ◽  
...  
Keyword(s):  
Expression Vector ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Homology Search ◽  
Protein Database ◽  
Gene Encoding ◽  
Reading Frame ◽  
Green Fluorescent

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

scholarly journals In Vivo Observations of Myosin II Dynamics Support a Role in Rear Retraction

1999 ◽  
Vol 10 (5) ◽  
pp. 1309-1323 ◽  
Author(s):  
Patricia A. Clow ◽  
James G. McNally
Keyword(s):  
Heavy Chain ◽  
Fluorescent Protein ◽  
Rotational Velocity ◽  
Myosin Ii ◽  
Cell Mass ◽  
Cell Movement ◽  
Posterior Cortex ◽  
Guidance Cues ◽  
Green Fluorescent

To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a “C,” which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC−) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC−), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC− cells, because these mutants exhibited proper rotational guidance cues. MHC− cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC− cells also exhibited mechanical defects in rotation, because they still moved aberrantly when seeded into wild-type mounds with proper rotational guidance cues. The mechanical defects in rotation may be mediated by the C-to-spot, because RLC− cells exhibited a defective C-to-spot, including a slower C-to-spot transition, consistent with this mutant’s slower rotational velocity.

scholarly journals Two-Step Assembly Dynamics of the Bacillussubtilis Divisome

2009 ◽  
Vol 191 (13) ◽  
pp. 4186-4194 ◽  
Author(s):  
Pamela Gamba ◽  
Jan-Willem Veening ◽  
Nigel J. Saunders ◽  
Leendert W. Hamoen ◽  
Richard A. Daniel
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Localization Pattern ◽  
Ftsz Ring ◽  
Time Lapse Microscopy ◽  
Green Fluorescent ◽  
Posttranscriptional Level

ABSTRACT Cell division in bacteria is carried out by about a dozen proteins which assemble at midcell and form a complex known as the divisome. To study the dynamics and temporal hierarchy of divisome assembly in Bacillus subtilis, we have examined the in vivo localization pattern of a set of division proteins fused to green fluorescent protein in germinating spores and vegetative cells. Using time series and time-lapse microscopy, we show that the FtsZ ring assembles early and concomitantly with FtsA, ZapA, and EzrA. After a time delay of at least 20% of the cell cycle, a second set of division proteins, including GpsB, FtsL, DivIB, FtsW, Pbp2B, and DivIVA, are recruited to midcell. Together, our data provide in vivo evidence for two-step assembly of the divisome. Interestingly, overproduction of FtsZ advances the temporal assembly of EzrA but not of DivIVA, suggesting that a signal different from that of FtsZ polymerization drives the assembly of late divisome proteins. Microarray analysis shows that FtsZ depletion or overexpression does not significantly alter the transcription of division genes, supporting the hypothesis that cell division in B. subtilis is mainly regulated at the posttranscriptional level.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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