J. Richard McIntosh

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at ≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 × 3.2 × 1.2 μm3) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455–476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, ≈66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells.

Abstract Nocodazole, the rapidly-reversible inhibitor of microtubule polymerization, has been used as a reagent to produce large numbers of mitotic mammalian cells at all stages of cell division. Mitotic cells are accumulated by incubation of cultures with 0.04 μg/ml of Nocodazole. Arrested cells are then harvested and resuspended in fresh medium where they progress through mitosis. HeLa, WI38, L929 and CHO cells proceed through cell division in a semi-synchronous manner following the removal of the drug. Nocodazole has very little effect on interphase metabolism, and following drug release, cells return to a normal cell cycle. Synchronization protocols and yields are presented.

Unstable Kinetochore-Microtubule Capture and Chromosomal Instability Following Deletion of CENP-E

A selective disruption of the mouse CENP-E gene was generated to test how this kinetochore-associated, kinesin-like protein contributes to chromosome segregation. The removal of CENP-E in primary cells produced spindles in which some metaphase chromosomes lay juxtaposed to a spindle pole, despite the absence of microtubules stably bound to their kinetochores. Most CENP-E-free chromosomes moved to the spindle equator, but their kinetochores bound only half the normal number of microtubules. Deletion of CENP-E in embryos led to early developmental arrest. Selective deletion of CENP-E in liver revealed that tissue regeneration after chemical damage was accompanied by aberrant mitoses marked by chromosome missegregation. CENP-E is thus essential for the maintenance of chromosomal stability through efficient stabilization of microtubule capture at kinetochores.

Force production by disassembling microtubules

Microtubules (MTs) are important components of the eukaryotic cytoskeleton: they contribute to cell shape and movement, as well as to the motions of organelles including mitotic chromosomes. MTs bind motor enzymes that drive many such movements, but MT dynamics can also contribute to organelle motility. Each MT polymer is a store of chemical energy that can be used to do mechanical work, but how this energy is converted to motility remains unknown. Here we show, by conjugating glass microbeads to tubulin polymers through strong inert linkages, such as biotin–avidin, that depolymerizing MTs exert a brief tug on the beads, as measured with laser tweezers. Analysis of these interactions with a molecular-mechanical model of MT structure and force production shows that a single depolymerizing MT can generate about ten times the force that is developed by a motor enzyme; thus, this mechanism might be the primary driving force for chromosome motion. Because even the simple coupler used here slows MT disassembly, physiological couplers may modulate MT dynamics in vivo.

Cytoplasmic dynein nomenclature

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

This work uses electron cryotomography to study the three-dimensional supramolecular organization of photosystem II and ATP synthase within the thylakoid membrane. It finds photosystem II as dimers in grana stacks, whereas ATP synthases are monomers located on minimally curved stromal thylakoids or grana end membranes but are absent from the highly curved grana margins, in clear contrast to the situation in mitochondria. We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.

Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching

Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo‐light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo‐EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo‐LM stage is that photobleaching is slower at cryogenic temperatures (−140°C) than at room temperature.

Spermatogenesis in males of the free-living nematode, Caenorhabditis elegans

We have studied the morphology of spermatogenesis in the free-living nematode Caenorhabditis elegans with the light and electron microscopes. The gonad of the adult male is a single, reflexed cylindrical structure containing all stages of spermatogenesis arranged in a single wave of development. The primary spermatocytes are at the end of the gonad most distal from its opening into the cloaca. In this region, the cells are syncytial, and there is a central core containing cytoplasm common to all the neighboring cells, an organization reminiscent of the ovary in the hermaphrodite. Moving toward the cloaca one encounters the stages of meiotic prophase. At diplotene and diakinesis the cells contain many Golgi complexes. Some of these Golgi complexes are associated with an urn-shaped vesicle with a dark amorphous collar about its neck. Other Golgi complexes are seen next to aggregates of microfilaments. These “fibrous bodies” become enveloped with a flattened vesicle that forms a boundary two membranes thick. After these two structures have grown in size, their membranes fuse to form a composite structure. The membranes at the site of fusion then develop dark-staining thickenings as they fold into convoluted sacs and tubes. At about this time, the cells go through the two meiotic divisions. At telophase II the composite structures and fibrous bodies cluster at the spindle poles and cleavage furrows not only separate the daughter cells, but they also slough off a substantial volume of cytoplasm. In the resulting spermatid the nucleus condenses to form the small mass of dark-staining chromatin characteristic of sperm. The microfilaments of the fibrous bodies now disappear while the membranes of the composite structures continue to fold. The texture of the cytoplasm becomes more dense and now contains numerous slender, wavy tubular elements. A part of each composite structure now fuses with the plasma membrane of the sperm to make an almost spherical invagination of extracellular space partially filled by the tortuous bits of membrane-bound cytoplasm formed by the foldings of the membranes of the composite structure.

Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans

During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.