The G-protein regulatory (GPR) motif-containing Leu–Gly–Asn-enriched protein (LGN) and Giα3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells

2006 ◽  
Vol 85 (12) ◽  
pp. 1233-1240 ◽  
Author(s):  
Joe B. Blumer ◽  
Ryoko Kuriyama ◽  
Thomas W. Gettys ◽  
Stephen M. Lanier
Keyword(s):  
G Protein ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Spindle Poles

scholarly journals Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

2002 ◽  
Vol 158 (5) ◽  
pp. 841-847 ◽  
Author(s):  
Marko J. Kallio ◽  
Victoria A. Beardmore ◽  
Jasminder Weinstein ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Anaphase Promoting Complex ◽  
Late Prophase ◽  
Checkpoint Signaling ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Late Telophase ◽  
Green Fluorescent

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

scholarly journals K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

2021 ◽  
Author(s):  
Marcus A Begley ◽  
April L Solon ◽  
Elizabeth Mae Davis ◽  
Michael Grant Sherrill ◽  
Ryoma Ohi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Binding Ability ◽  
Microtubule Binding ◽  
Mechanical Integrity ◽  
Spindle Poles ◽  
Rigor State ◽  
Physical Response

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors. [Media: see text] [Media: see text] [Media: see text]

scholarly journals TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

2011 ◽  
Vol 195 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Nan Ma ◽  
Janel Titus ◽  
Alyssa Gable ◽  
Jennifer L. Ross ◽  
Patricia Wadsworth
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Spindle Assembly ◽  
Artificial Chromosome ◽  
Fiber Formation ◽  
Interaction Domain ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Multiple Spindle ◽  
And Function

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2–Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.

The Role of the Centrosome in Development and Progression of Breast Cancer

2001 ◽  
Vol 7 (S2) ◽  
pp. 582-583
Author(s):  
W. Lingle ◽  
J. Salisbury ◽  
S. Barrett ◽  
V. Negron ◽  
C. Whitehead
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Microtubule Organizing Center ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Close Proximity ◽  
Interphase Cells ◽  
Organizing Center

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

scholarly journals Mechanical and genetic separation of aster- and midzone-positioned cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 381-383 ◽  
Author(s):  
Henrik Bringmann
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mitotic Spindle ◽  
Heterotrimeric G Proteins ◽  
Contractile Ring ◽  
Spindle Poles ◽  
Spindle Midzone ◽  
Spindle Microtubules ◽  
Two Populations ◽  
Provided Examples

The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.

Endosome positioning during cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 442-443 ◽  
Author(s):  
Guillaume Montagnac ◽  
Philippe Chavrier
Keyword(s):  
Targeted Delivery ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Recycling Endosomes ◽  
Spindle Poles

In mammalian cells, completion of cytokinesis relies on targeted delivery of recycling membranes to the midbody. At this step of mitosis, recycling endosomes are organized as clusters located at the mitotic spindle poles as well as at both sides of the midbody. However, the mechanism that controls endosome positioning during cytokinesis is not known. Here, we discuss the possible mechanisms that drive the formation of endosomal clusters and the importance of this process for the targeted delivery of recycling membranes to the midbody.

scholarly journals Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

2021 ◽  
Author(s):  
Alessandro Dema ◽  
Jeffrey van Haren ◽  
Torsten Wittmann
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Live Cells ◽  
Cell Cortex ◽  
Reversible Inactivation ◽  
Molecular Systems ◽  
Spindle Poles ◽  
Spindle Dynamics ◽  
Spindle Function ◽  
Transient Relaxation

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, π-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

scholarly journals A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes

Open Biology ◽  
2018 ◽  
Vol 8 (7) ◽  
pp. 170218 ◽  
Author(s):  
Jane Harmer ◽  
Katie Towers ◽  
Max Addison ◽  
Sue Vaughan ◽  
Michael L. Ginger ◽  
...  
Keyword(s):  
Basal Body ◽  
Mitotic Spindle ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Molecular Level ◽  
African Trypanosomes ◽  
Spindle Poles ◽  
Microtubule Organizing Centres ◽  
Polar Spindle ◽  
And Function

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, Tb FOPL and Tb OFD1, in the evolutionarily divergent sleeping sickness parasite Trypanosoma brucei , showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, Tb FOPL, affects neither axoneme nor flagellum elongation. Instead, Tb FOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar Tb FOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, Tb FOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for Tb FOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of Tb FOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

scholarly journals The mitotic spindle mediates inheritance of the Golgi ribbon structure

2009 ◽  
Vol 184 (3) ◽  
pp. 391-397 ◽  
Author(s):  
Jen-Hsuan Wei ◽  
Joachim Seemann
Keyword(s):  
Mitotic Spindle ◽  
Daughter Cell ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Golgi Membranes ◽  
Ribbon Structure ◽  
Golgi Ribbon

The mammalian Golgi ribbon disassembles during mitosis and reforms in both daughter cells after division. Mitotic Golgi membranes concentrate around the spindle poles, suggesting that the spindle may control Golgi partitioning. To test this, cells were induced to divide asymmetrically with the entire spindle segregated into only one daughter cell. A ribbon reforms in the nucleated karyoplasts, whereas the Golgi stacks in the cytoplasts are scattered. However, the scattered Golgi stacks are polarized and transport cargo. Microinjection of Golgi extract together with tubulin or incorporation of spindle materials rescues Golgi ribbon formation. Therefore, the factors required for postmitotic Golgi ribbon assembly are transferred by the spindle, but the constituents of functional stacks are partitioned independently, suggesting that Golgi inheritance is regulated by two distinct mechanisms.

scholarly journals Functional expression and characterization of the C. elegans G-protein-coupled FLP-2 Receptor (T19F4.1) in mammalian cells and yeast

2013 ◽  
Vol 3 ◽  
pp. 1-7 ◽  
Author(s):  
Martha J. Larsen ◽  
Elizabeth Ruiz Lancheros ◽  
Tracey Williams ◽  
David E. Lowery ◽  
Timothy G. Geary ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Functional Expression ◽  
C Elegans ◽  
G Protein Coupled
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