Biomedical subjects

Mark S Ladinsky

Publications and source records attributed to Mark S Ladinsky.

5 recordsLinked to original sources

PubMed2006

Vitreous cryo-sectioning of cells facilitated by a micromanipulator.

Sectioning vitrified cells and tissues for cryo-electron microscopy is more challenging than room-temperature sectioning of plastic-embedded samples. As the sample must be kept very cold (<-130 degrees C) and because there is no liquid upon which the sections can float as they are cut, transferring the sections from the knife edge to a grid is one of the more difficult steps in the process. We employed a micromanipulator to hold and control the cryo-sections as they come off the knife. This allows slower cutting speeds than are typically used in vitreous cryo-sectioning and contributes to better control during cutting, which facilitates repeatable placement of a ribbon of sections onto a grid. The ribbon is kept under tension during the entire cutting process, which may decrease folding and/or compression, features that are inherent to vitreous sections. Furthermore, the added control afforded by this technique makes it easier for multiple ribbons to be placed on a single grid, thereby increasing the number of sections that can be examined and imaged during a microscopy session. It even allows for serial cryo-electron microscopy. As such, this approach is an advance in the cryo-microtomy of vitreous sections.

Cryoelectron Microscopy↗

PubMed2005

GMx33 associates with the trans-Golgi matrix in a dynamic manner and sorts within tubules exiting the Golgi.

The trans-Golgi matrix consists of a group of proteins dynamically associated with the trans-Golgi and thought to be involved in anterograde and retrograde Golgi traffic, as well as interactions with the cytoskeleton and maintenance of the Golgi structure. GMx33 is localized to the cytoplasmic face of the trans-Golgi and is also present in a large cytoplasmic pool. Here we demonstrate that GMx33 is dynamically associated with the trans-Golgi matrix, associating and dissociating with the Golgi in seconds. GMx33 can be locked onto the trans-Golgi matrix by GTPgammaS, indicating that its association is regulated in a GTP-dependent manner like several other Golgi matrix proteins. Using live-cell imaging we show that GMx33 exits the Golgi associated with tubules and within these tubules GMx33 segregates from transmembrane proteins followed by fragmentation of the tubules into smaller tubules and vesicles. Within vesicles produced by an in vitro budding reaction, GMx33 remains segregated in a matrixlike tail region that sometimes contains Golgin-245. This trans-matrix often links a few vesicles together. Together these data suggest that GMx33 is a member of the trans-Golgi matrix and offer clues regarding the role of the trans-Golgi matrix in sorting and exit from the Golgi.

Adenosine Triphosphate↗

PubMed2004

Predicting function from structure: 3D structure studies of the mammalian Golgi complex.

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

Animals↗

PubMed2002

Three-dimensional reconstruction of cytoplasmic membrane networks in parietal cells.

There is general agreement that stimulation and consequent secretion of gastric parietal cells result in a great expansion of the apical canalicular membrane at the expense of an extensive intracellular network of membranes rich in the gastric proton pump (H,K-ATPase). However, there is ongoing controversy as to the precise nature of the intracellular membrane network, conventionally called tubulovesicles. At the heart of this controversy lies the question of whether tubulovesicles are a distinct membrane compartment or whether they are continuous with the apical plasma membrane. To address this controversy we used high-pressure, rapid freezing techniques to fix non-stimulated (resting) rabbit gastric glands for electron microscopy. Ultra-thin (60-70 nm) serial sections were used for conventional TEM; 400-500 nm sections were used for tomography. Images were digitized and models constructed using Midas and Imod software (http://bio3d.colorado.edu ). Images were aligned and contours drawn on specific cellular structures. The contours from a stack of serial sections were arranged into objects and meshed into 3D structures. For resting parietal cells our findings are as follows: (1) The apical canaliculus is a microvilli-decorated, branching membrane network that extends into and throughout the parietal cell. This agrees well with a host of previous studies. (2) The plentiful mitochondria form an extensive reticular network throughout the cytoplasm. This has not previously been reported for the parietal cell, and the significance of this observation and the dynamics of the mitochondrial network remain unknown. (3) H,K-ATPase-rich membranes do include membrane tubules and vesicles; however, the tubulovesicular compartment is chiefly comprised of small stacks of cisternae. Thus a designation of tubulocisternae seems appropriate; however, in the resting cell there are no continuities between the apical canaliculus and the tubulocisternae or between tubulocisternae. These data support the recruitment-recycling model of parietal cell stimulation.

Animals↗

PubMed2002

Structure of the Golgi and distribution of reporter molecules at 20 degrees C reveals the complexity of the exit compartments.

Incubating cells at 20 degrees C blocks transport out of the Golgi complex and amplifies the exit compartments. We have used the 20 degrees C block, followed by EM tomography and serial section reconstruction, to study the structure of Golgi exit sites in NRK cells. The dominant feature of Golgi structure in temperature-blocked cells is the presence of large bulging domains on the three trans-most cisternae. These domains extend laterally from the stack and are continuous with "cisternal" domains that maintain normal thickness and alignment with the other stacked Golgi cisternae. The bulging domains do not resemble the perpendicularly extending tubules associated with the trans-cisternae of control cells. Such tubules are completely absent in temperature-blocked cells. The three cisternae with bulging domains can be identified as trans by their association with specialized ER and the presence of clathrin-coated buds on the trans-most cisterna only. Immunogold labeling and immunoblots show a significant degradation of a medial- and a trans-Golgi marker with no evidence for their redistribution within the Golgi or to other organelles. These data suggest that exit from the Golgi occurs directly from three trans-cisternae and that specialized ER plays a significant role in trans-Golgi function.

Animals↗