No abstract

Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for the way dynein generates force to slide microtubules. They also reveal two dynein linkers that may provide "hard-wiring" to coordinate motor enzyme action, both circumferentially and along the axoneme. Periodic densities were also observed inside doublet microtubules; these may contribute to doublet stability.

Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at ∼7-nm resolution. The reconstruction shown here (∼1 × 1 × 4 μm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled “wells” open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 μm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.