Drosophila S2 Cells as a Model System to Investigate Mitotic Spindle Dynamics, Architecture, and Function

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end–directed motor proteins. Here, we have characterized the roles of two minus end–directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end–tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end–directed motors cooperate to ensure spindle pole coalescence during mitosis.

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Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

The Journal of Cell Biology ◽

10.1083/jcb.200407090 ◽

2004 ◽

Vol 167 (5) ◽

pp. 831-840 ◽

Cited By ~ 199

Author(s):

Helder Maiato ◽

Conly L. Rieder ◽

Alexey Khodjakov

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Spindle Pole ◽

S2 Cells ◽

Spindle Poles ◽

Drosophila S2 Cells ◽

Mitotic Spindle Assembly ◽

Normal Mitosis ◽

Proper Orientation ◽

Dependent Mechanism

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

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Anaphase B spindle dynamics in Drosophila S2 cells: Comparison with embryo spindles

Cell Division ◽

10.1186/1747-1028-6-8 ◽

2011 ◽

Vol 6 (1) ◽

pp. 8 ◽

Cited By ~ 10

Author(s):

Jane de Lartigue ◽

Ingrid Brust-Mascher ◽

Jonathan M Scholey

Keyword(s):

S2 Cells ◽

Spindle Dynamics ◽

Drosophila S2 Cells ◽

Anaphase B

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dGRASP Localization and Function in the Early Exocytic Pathway in Drosophila S2 Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0938 ◽

2005 ◽

Vol 16 (9) ◽

pp. 4061-4072 ◽

Cited By ~ 67

Author(s):

Vangelis Kondylis ◽

Kirsten M. Spoorendonk ◽

Catherine Rabouille

Keyword(s):

Structural Integrity ◽

De Novo ◽

Matrix Proteins ◽

S2 Cells ◽

Anterograde Transport ◽

Golgi Stack ◽

Drosophila S2 Cells ◽

And Function ◽

Exocytic Pathway ◽

Golgi Matrix

The de novo model for Golgi stack biogenesis predicts that membrane exiting the ER at transitional ER (tER) sites contains and recruits all the necessary molecules to form a Golgi stack, including the Golgi matrix proteins, p115, GM130, and GRASP65/55. These proteins leave the tER sites faster than Golgi transmembrane resident enzymes, suggesting that they act as a template nucleating the formation of the Golgi apparatus. However, the localization of the Golgi matrix proteins at tER sites is only shown under conditions where exit from the ER is blocked. Here, we show in Drosophila S2 cells, that dGRASP, the single Drosophila homologue of GRASP65/55, localizes both to the Golgi membranes and the tER sites at steady state and that the myristoylation of glycine 2 is essential for the localization to both compartments. Its depletion for 96 h by RNAi gave an effect on the architecture of the Golgi stacks in 30% of the cells, but a double depletion of dGRASP and dGM130 led to the quantitative conversion of Golgi stacks into clusters of vesicles and tubules, often featuring single cisternae. This disruption of Golgi architecture was not accompanied by the disorganization of tER sites or the inhibition of anterograde transport. This shows that, at least in Drosophila, the structural integrity of the Golgi stacks is not required for efficient transport. Overall, dGRASP exhibits a dynamic association to the membrane of the early exocytic pathway and is involved in Golgi stack architecture.

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Automated mitotic spindle tracking suggests a link between spindle dynamics, spindle orientation, and anaphase onset in epithelial cells

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0355 ◽

2017 ◽

Vol 28 (6) ◽

pp. 746-759 ◽

Cited By ~ 14

Author(s):

Matthew E. Larson ◽

William M. Bement

Keyword(s):

Mitotic Spindle ◽

Spindle Positioning ◽

Tissue Organization ◽

Anaphase Onset ◽

Spindle Dynamics ◽

And Function ◽

Spindle Position ◽

The Relationship ◽

Rotational Oscillations ◽

Final Orientation

Proper spindle positioning at anaphase onset is essential for normal tissue organization and function. Here we develop automated spindle-tracking software and apply it to characterize mitotic spindle dynamics in the Xenopus laevis embryonic epithelium. We find that metaphase spindles first undergo a sustained rotation that brings them on-axis with their final orientation. This sustained rotation is followed by a set of striking stereotyped rotational oscillations that bring the spindle into near contact with the cortex and then move it rapidly away from the cortex. These oscillations begin to subside soon before anaphase onset. Metrics extracted from the automatically tracked spindles indicate that final spindle position is determined largely by cell morphology and that spindles consistently center themselves in the XY-plane before anaphase onset. Finally, analysis of the relationship between spindle oscillations and spindle position relative to the cortex reveals an association between cortical contact and anaphase onset. We conclude that metaphase spindles in epithelia engage in a stereotyped “dance,” that this dance culminates in proper spindle positioning and orientation, and that completion of the dance is linked to anaphase onset.

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