Martin Srayko

Cortical Microtubule Contacts Position the Spindle in C. elegans Embryos

Interactions between microtubules and the cell cortex play a critical role in positioning organelles in a variety of biological contexts. Here we used Caenorhabditis elegans as a model system to study how cortex-microtubule interactions position the mitotic spindle in response to polarity cues. Imaging EBP-2::GFP and YFP::alpha-tubulin revealed that microtubules shrink soon after cortical contact, from which we propose that cortical adaptors mediate microtubule depolymerization energy into pulling forces. We also observe association of dynamic microtubules to form astral fibers that persist, despite the catastrophe events of individual microtubules. Computer simulations show that these effects, which are crucially determined by microtubule dynamics, can explain anaphase spindle oscillations and posterior displacement in 3D.

Katanin Disrupts the Microtubule Lattice and Increases Polymer Number in C. elegans Meiosis

Katanin is a heterodimer that exhibits ATP-dependent microtubule-severing activity in vitro. In Xenopus egg extracts, katanin activity correlates with the addition of cyclin B/cdc2, suggesting a role for microtubule severing in the disassembly of long interphase microtubules as the cell prepares for mitosis. However, studies from plant cells, cultured neurons, and nematode embryos suggest that katanin could be required for the organization or postnucleation processing of microtubules, rather than the dissolution of microtubule structures. Here we reexamine katanins role by studying acentrosomal female meiotic spindles in C. elegans embryos. In mutant embryos lacking katanin, microtubules form around meiotic chromatin but do not organize into bipolar spindles. By using electron tomography, we found that katanin converts long microtubule polymers into shorter microtubule fragments near meiotic chromatin. We further show that turning on katanin during mitosis also creates a large pool of short microtubules near the centrosome. Furthermore, the identification of katanin-dependent microtubule lattice defects supports a mechanism involving an initial perforation of the protofilament wall. Taken together, our data suggest that katanin is used during meiotic spindle assembly to increase polymer number from a relatively inefficient chromatin-based microtubule nucleation pathway.

Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis

Publisher Summary The early embryo of Caenorhabditis elegans is one of the most powerful model systems to study cell division. This chapter develops a correlative light and electron microscopic approach to stage early C. elegans embryos prior to high-pressure freezing and electron tomography. Cellulose microcapillaries are used to contain the embryos in short transparent tubes. These permit the viewing of early mitotic events under a light microscope. Next step is to stage selected embryos by light microscopy (LM), to immobilize these staged embryos by high-pressure freezing, and to fix them by freeze-substitution. The chapter describes the thin-layer embedding and 3D reconstruction of spindle components by electron tomography. Using the newly developed rapid transfer system (RTS) of the Leica EMPACT2 high-pressure freezer reduces the time between light microscopic observation and cryoimmobilization to about 5 sec. The correlative approach allows systematic structure-function studies in staged early wild-type embryos and those treated with RNA-mediated interference (RNAi) to reduce the level of specific gene products.

Caenorhabditis elegans TAC-1 and ZYG-9 Form a Complex that Is Essential for Long Astral and Spindle Microtubules

TACC (transforming acidic coiled-coil) proteins were first identified by their ability to transform cell lines [1], and links between human cancer and the overexpression of TACC proteins highlight the importance of understanding the biological function of this family of proteins. Herein, we describe the characterization of a new member of the TACC family of proteins in Caenorhabditis elegans, TAC-1. In other systems, TACC proteins associate with the XMAP215 family of microtubule-stabilizing proteins; however, it is unclear whether TACC proteins have microtubule-based functions distinct from XMAP215. We depleted both the XMAP215 ortholog ZYG-9 and TAC-1 via dsRNA-mediated interference (RNAi). We found that tac-1(RNAi) resulted in microtubule-based defects that were very similar to zyg-9(RNAi). Furthermore, TAC-1 and ZYG-9 are required for long astral microtubules in general and long spindle microtubules during spindle assembly. Loss of either protein did not affect the alpha-tubulin immunofluorescence intensity near centrosomes; this finding suggests that microtubule nucleation was not compromised. Both proteins localize to centrosomes and the kinetochore/microtubule region of chromosomes in metaphase and early anaphase. Furthermore, we found that ZYG-9 and TAC-1 physically interact in vivo, and this interaction is important for the efficient localization of the ZYG-9/TAC-1 complex to centrosomes.

The C. elegans RSA Complex Localizes Protein Phosphatase 2A to Centrosomes and Regulates Mitotic Spindle Assembly

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.

Electron microscopy of the early Caenorhabditis elegans embryo

The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser‐induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time‐resolved fixation to arrest development at specific stages. The second method uses high‐pressure freezing of whole worms followed by freeze‐substitution (HPF‐FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high‐resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF‐FS and electron tomography. This procedure combines the advantages of time‐resolved fixation and superior ultrastructural preservation by high‐pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.