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

Abstract

In order to perpetuate their genetic content, eukaryotic cells have developed a microtubule-based machine known as the mitotic spindle. Independently of the system studied, mitotic spindles share at least one common characteristic -the dynamic nature of microtubules. This property allows the constant plasticity needed to assemble a bipolar structure, make proper kinetochoremicrotubule attachments, segregate chromosomes and finally disassemble the spindle and reform an interphase microtubule array. Here we describe a variety of experimental approaches currently used in our laboratory to study microtubule dynamics during mitosis using Drosophila melanogaster S2 cells as a model. By using quantitative live-cell imaging microscopy in combination with an advantageous labeling background, we illustrate how several cooperative pathways are used to build functional mitotic spindles. We illustrate different ways of perturbing spindle microtubule dynamics, including pharmacological inhibition and RNA interference of proteins that directly or indirectly impair microtubule dynamics. Additionally, we demonstrate the advantage of using fluorescent speckle microscopy (FSM) to investigate an intrinsic property of spindle microtubules known as poleward flux. Finally, we developed a set of laser microsurgery-based experiments that allow, with unique spatiotemporal resolution, the study of specific spindle-structures (e.g. centrosomes, microtubules and kinetochores) and their respective roles during mitosis.3

Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species

“…Because the starting S2 cells are polyploid and are known to be highly proliferative to begin with (Moutinho-Pereira et al, 2010), we wondered if these properties could have contributed to the induction process. Thus we tried another Drosophilia cell line, BG2, which is derived from the central nervous system and which is less proliferative (Ui-Tei et al, 2000).…”

“…Second, proliferation was enhanced, but the starting S2 cells were already relatively proliferative. This difference might be because S2 cells are partially differentiated, aneuploid, renewable cells (Moutinho-Pereira et al, 2010). As such, these cells are a work-horse type of cell line, such as HeLa cells for humans, more so than true primary somatic cells.…”

A kinesin Klp10A mediates cell cycle-dependent shuttling of Piwi between nucleus and nuage

“…To this end, we first examined the localization of Piwi and Vasa in control vs. klp10A RNAi GSCs/SGs. We further detected physical interaction and colocalization between Vasa and Piwi specifically when cells were blocked in metaphase with MG132 (a proteasome inhibitor that arrests cells in mitosis [52]), or with colcemid (S9 Fig) [31]. These results suggest that Piwi gains access to nuage specifically during mitosis.…”

Cell Signaling Experiments Driven by Optical Manipulation

“…Several laboratories use laser dissection systems as a complementary approach to pharmacological and genetic inhibition of single proteins because they allow selective manipulation of whole organelles and structures with high spatial resolution [106]. However, one of the more challenging applications of laser dissection is the study of wound healing and the regeneration processes of injured tissues.…”