scholarly journals Direct observation of branching MT nucleation in living animal cells

2019 ◽  
Author(s):  
Vikash Verma ◽  
Thomas J. Maresca
Keyword(s):  
Direct Observation ◽  
Branch Point ◽  
Living Animal ◽  
Nucleation Event ◽  
Animal Cells ◽  
Entire Process ◽  
Rhoa Activation ◽  
Branch Angle ◽  
Nucleation Mechanisms ◽  
Ring Complex

ABSTRACTCentrosome-mediated microtubule (MT) nucleation has been well-characterized; however, numerous non-centrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well-conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multi-color TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells. The stereotypical branching nucleation event entailed augmin first binding to a “mother” MT, recruitment of γ-TuRC after 16s, followed by nucleation 15s later of a “daughter” MT at a 36° branch angle. Daughters typically remained attached throughout their ~40s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require D-TPX2 (Drosophila TPX2) or evident regulation by a RanGTP gradient, enhanced localized RhoA activation during cytokinesis.

scholarly journals Direct observation of branching MT nucleation in living animal cells

2019 ◽  
Vol 218 (9) ◽  
pp. 2829-2840 ◽  
Author(s):  
Vikash Verma ◽  
Thomas J. Maresca
Keyword(s):  
Direct Observation ◽  
Branch Point ◽  
Living Animal ◽  
Nucleation Event ◽  
Animal Cells ◽  
Rhoa Activation ◽  
Branch Angle ◽  
Nucleation Mechanisms ◽  
Ring Complex ◽  
Drosophila Cells

Centrosome-mediated microtubule (MT) nucleation has been well characterized; however, numerous noncentrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multicolor TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells during anaphase. The steps of a stereotypical branching nucleation event entailed augmin binding to a mother MT and recruitment of γ-TuRC after 15 s, followed by nucleation 16 s later of a daughter MT at a 36° branch angle. Daughters typically remained attached throughout their ∼40-s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require Drosophila TPX2, enhanced localized RhoA activation during cytokinesis.

scholarly journals The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control

2021 ◽  
Author(s):  
Xiaolei Gao ◽  
Saturnino Herrero ◽  
Valentin Wernet ◽  
Sylvia Erhardt ◽  
Oliver Valerius ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Cell Types ◽  
Spindle Pole ◽  
Receptor Protein ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Spindle Pole Bodies ◽  
Organizing Center ◽  
Ring Complex ◽  
Core Components

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) were described in many cell types. Functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans additional MTOCs were discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates SPB outer plaque with sMTOC activities. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest polo-like kinase as a regulator of MTOC activities and as a scaffolding unit through interaction with γ-tubulin ring complex receptors.

scholarly journals Translation of plant-specific messenger RNAs in living animal cells

FEBS Letters ◽  
1977 ◽  
Vol 81 (1) ◽  
pp. 10-12 ◽  
Author(s):  
J. Schröder ◽  
F. Kreuzaler ◽  
J. Schmock
Keyword(s):  
Messenger Rnas ◽  
Living Animal ◽  
Animal Cells

scholarly journals Characterization of a Drosophila Centrosome Protein CP309 That Shares Homology with Kendrin and CG-NAP

2004 ◽  
Vol 15 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Shin-ichi Kawaguchi ◽  
Yixian Zheng
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Biochemical Studies ◽  
Ring Complex ◽  
Centrosome Protein

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the γ-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the γ-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.

Dicistronic selection for nuclear proteins in living animal cells

Gene ◽  
1993 ◽  
Vol 137 (1) ◽  
pp. 145-149 ◽  
Author(s):  
an Smarda ◽  
Joseph S. Lipsick
Keyword(s):  
Nuclear Proteins ◽  
Living Animal ◽  
Animal Cells ◽  
Selection For

Nondestructive micropatterning of living animal cells using focused femtosecond laser-induced impulsive force

2007 ◽  
Vol 91 (2) ◽  
pp. 023904 ◽  
Author(s):  
Takahiro Kaji ◽  
Syoji Ito ◽  
Hiroshi Miyasaka ◽  
Yoichiroh Hosokawa ◽  
Hiroshi Masuhara ◽  
...  
Keyword(s):  
Femtosecond Laser ◽  
Living Animal ◽  
Impulsive Force ◽  
Animal Cells

Plant and animal profilins are functionally equivalent and stabilize microfilaments in living animal cells

1996 ◽  
Vol 109 (1) ◽  
pp. 83-90 ◽  
Author(s):  
M. Rothkegel ◽  
O. Mayboroda ◽  
M. Rohde ◽  
C. Wucherpfennig ◽  
R. Valenta ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Laser Scanning ◽  
Birch Pollen ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Kinetic Properties ◽  
Living Animal ◽  
Animal Cells ◽  
Microfilament Bundles ◽  
The Stability

We have analyzed the degree of functional similarity between birth and mammalian profilins, two members of the profilin family which show only a moderate sequence homology (22%) in living animal cells. The plant profilin, derived from birch pollen, was stably expressed in BHK-21 cells. Plant and endogenous profilin synthesis and cellular distribution were monitored by specific monoclonal antibodies. Quantitation of profilin and actin on calibrated immunoblots showed that two stable clones contained in total 1.4 and 2.0 times as much profilin as the parental cells. Using double fluorescence and confocal laser scanning microscopy, it was seen that the endogenous and the plant profilin colocalized with dynamic microfilaments, in particular with F-actin-rich foci and cortical microfilament webs of spreading cells, with dynamic microfilament bundles induced by serum deprival, and with cytochalasin D- and latrunculin-induced transient F-actin aggregates. The increase in the overall profilin concentration correlated with a significantly higher resistance of actin filaments to these drugs. Our data indicate that even profilins of highly distant evolutionary origin can functionally substitute for each other and support the hypothesis that in animal cells, profilins are engaged in regulating either the stability or the kinetic properties of actin filaments.

scholarly journals Case studies of particle formation events observed in boreal forests: implications for nucleation mechanisms

2008 ◽  
Vol 8 (2) ◽  
pp. 5683-5723 ◽  
Author(s):  
F. Yu ◽  
R. Turco
Keyword(s):  
Case Studies ◽  
Boreal Forests ◽  
Forest Site ◽  
Nucleation Event ◽  
Air Masses ◽  
Size Dependent ◽  
Stable Clusters ◽  
Nucleation Mechanisms ◽  
Total Event ◽  
Physical And Chemical

Abstract. Aerosol nucleation events observed worldwide may have significant climatic and health implications. However, the specific nucleation mechanisms remain ambiguous. Here, we report case studies of six nucleation events observed during an intensive field campaign at a boreal forest site (Hyytiälä, Finland) in spring 2005. The present analysis is based on comprehensive kinetic simulations using an ion-mediated nucleation (IMN) model in which the key physical and chemical parameters are constrained by a variety of recent measurements. Out of roughly 30 nucleation event days sampled during the campaign, four were initially selected on the basis of indications that the observed air masses were relatively homogeneous. It happens that all four of these days exhibited medium to high electrical overcharging of the nucleated nanoparticles. In each of these well-defined cases, reasonable agreement is found between the predictions and field data for a range of variables, including critical nucleation sizes, size-dependent overcharging ratios, and the concentrations of 1.8–3 nm stable clusters and 3–6 nm particles, and their diurnal variations. However, to extend the scope of the study, one case of weak electrical overcharging, and one of clear undercharging, of the nucleated particles were also selected. These electrical states represented less than about 20% of the total event-days recorded, and among this smaller sample there were no days on which the sampled air masses appeared reasonable uniform over the entire nucleation event. Thus, it is perhaps not surprising that the consistency between model simulations and measurements during these more anomalous periods was less satisfying. We tentatively conclude that the outcomes in these cases were influenced by, among other things, the significant variability in the sampled air masses and the possible role of species other than sulfuric acid in the nucleation process. Statistically, roughly 80% of the nucleation events recorded during the Hyytiälä campaign exhibited mean size-dependent overcharging ratios within the range, or exceeding, those predicted by the IMN model, suggesting that ion nucleation processes are significant. The nucleation rates calculated using the IMN modeling approach are contrasted with those predicted by other theories/models, and key differences between the results are discussed.

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