David J. Sharp

Microtubule motors in mitosis

The mitotic spindle uses microtubule-based motor proteins to assemble itself and to segregate sister chromatids. It is becoming clear that motors invoke several distinct mechanisms to generate the forces that drive mitosis. Moreover, in carrying out its function, the spindle appears to pass through a series of transient steady-state structures, each established by a delicate balance of forces generated by multiple complementary and antagonistic motors. Transitions from one steady state to the next can occur when a change in the activity of a subset of mitotic motors tips the balance.

Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.

Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.

The positioning of centrosomes, or microtubule-organizing centres, within cells plays a critical part in animal development. Here we show that, in Drosophila embryos undergoing mitosis, the positioning of centrosomes within bipolar spindles and between daughter nuclei is determined by a balance of opposing forces generated by a bipolar kinesin motor, KLP61F, that is directed to microtubule plus ends, and a carboxy-terminal kinesin motor, Ncd, that is directed towards microtubule minus ends. This activity maintains the spacing between separated centrosomes during prometaphase and metaphase, and repositions centrosomes and daughter nuclei during late anaphase and telophase. Surprisingly, we do not observe a function for KLP61F in the initial separation of centrosomes during prophase. Our data indicate that KLP61F and Ncd may function by crosslinking and sliding antiparallel spindle microtubules in relation to one another, allowing KLP61F to push centrosomes apart and Ncd to pull them together.

Mitosis, microtubules, and the matrix

The mechanical events of mitosis depend on the action of microtubules and mitotic motors, but whether these spindle components act alone or in concert with a spindle matrix is an important question.

Transmission electron microscopy of thin sections of Drosophila: Preparation of embryos using n-heptane and glutaraldehyde

This protocol describes the simultaneous permeabilization of Drosophila embryos with n-heptane and initial fixation with glutaraldehyde. Even though the vitelline membrane around the embryo is chemically permeabilized, it must be manually removed to achieve infiltration with embedding resins. Once the embryo is embedded, it can be sectioned for transmission electron microscopy (TEM). This procedure can produce excellent preservation for ultrastructural analysis, and is useful for situations where optimal preservation (e.g., by high-pressure freezing) is not required or is not feasible.

Preparation of Drosophila Specimens for Examination by Transmission Electron Microscopy

There is no single, simple procedure for fixing and embedding all tissues for transmission electron microscopy (TEM). The chemistry of different cell types is to some extent unique, and this affects the way each cell type reacts to the wide array of fixatives, buffers, organic solvents, and resins used in TEM specimen preparation. A recurring theme in those organisms or cell types that are difficult to fix is the presence of a diffusion barrier that prevents the free diffusion of fixative and other chemicals in and out of the cell or tissue. This in turn means that fixation takes a relatively long time (measured in minutes or tens of minutes in some cases), during which the cells begin autolysis or are otherwise degraded from their original state. Drosophila requires specific preparation methods for TEM because most fly tissues are surrounded by significant diffusion barriers. In the embryo, it is the vitelline envelope, and in larvae and adults, it is the cuticle. In this article, we discuss methods that have evolved to cope with these barriers to achieve reasonable preservation of ultrastructure.

Postembedding Immunolabeling of Thin Sections of Drosophila Tissues for Transmission Electron Microscopy

Postembedding immunolabeling using resin sections is the recommended method for beginners carrying out electron microscopy (EM) immunolabeling. Postembedding labeling refers to labeling on sections, which is a method of gaining access to the interior of the cell without the harshness of detergent or ionic extraction as is performed with preembed labeling. Investigators already familiar with routine EM-sectioning techniques find EM immunolabeling using resin sections easiest to do, as procedures are similar to those used when performing light microscopy (LM) immunolabeling, but using a different resin. In addition, the overall preservation of structure is best in resin compared to use of cryosections or preembed labeling. The most critical component of immunoEM (iEM) is what primary antibody to use. This protocol descibes antibody labeling procedures for postembedding iEM using thin sections of Drosophila tissues.

Immunolabeling of thin sections of Drosophila tissues for transmission electron microscopy

The main advantage of electron microscopy (EM) for immunolabeling is resolution, but there is also another aspect that is often overlooked. For many investigators, the definitive image of an organelle is the one generated by EM. This is especially true for membranous organelles, with the possible exception of the nucleus and plant vacuoles. For example, references to the Golgi apparatus, smooth and rough endoplasmic reticulum, centriole, kinetochore, or mitochondrion typically bring to mind the images in an electron micrograph. The components of the cytoskeleton also have characteristic structural features that are associated with their EM image. Thus, it can be more effective for investigators to view gold particles superimposed over the image of a microtubule (MT) or mitochondrion using EM than to see bright dots or lines in the light microscope. This is especially true if the immunofluorescence image is of fixed cells. Here, we provide an overview of methods of EM immunolabeling used for localizing specific antigens on thin sections of Drosophila tissues.

Transmission electron microscopy of thin sections of Drosophila: Conventional chemical fixation of embryos using trialdehyde

High-pressure freezing (HPF) followed by freeze-substitution is a valuable method for specimen preservation for transmission electron microscopy (TEM) in Drosophila. However, not all projects require this level of precision. In addition, some tissues are too large to fit into the HPF specimen carriers, and some fly tissues such as eyes and ovaries do not freeze well. This protocol describes a trialdehyde fixation procedure for embryos, to be used in situations where optimal preservation is not required or when HPF is not an option. Because the vitelline membrane is impermeable to aqueous solvents, it is necessary to either mechanically disrupt it or render it permeable by treatment with organic solvents. Good ultrastructural preservation has been achieved by puncturing embryos immersed in fixative with extremely sharp tungsten needles, as described here.