Metallothionein as a clonable tag for protein localization by electron microscopy of cells.

A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.

Silver enhancement of Nanogold particles during freeze substitution for electron microscopy.

Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well-fixed, well-resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno-localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron-dense markers, like Nanogold(R), will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor-mediated membrane traffic.

The use of filter membranes for high-pressure freezing of cell monolayers.

Rapid freezing of cells and tissues, followed by freeze-substitution fixation and plastic embedding, has become a highly reliable method for preparing samples for imaging in the electron microscope. High-pressure freezing is an efficient means of immobilizing suspensions of yeasts, thick pellets of mammalian cells, or small (< 0.5 mm) pieces of plant or animal tissue. Monolayers of cultured mammalian cells that are too thick for efficient immobilization by other modes of rapid freezing have also been successfully preserved by this method. Monolayer cultures are often important because they can be imaged by light microscopy (LM) both before and after their preparation for electron microscopy (EM). Additionally, some monolayer cultures serve as model systems for physiological processes, so it is important that cells under study can grow on a substrate that is both physiologically appropriate and convenient for EM processing. Here we describe a reliable method for preparing mammalian cell monolayers (PtK1 and polarized MDCK) for EM. Our protocol results in good preservation of cellular ultrastructure, it is a useful companion to studies of cell physioloy and, with some limitation, is suitable for correlative LM and EM.

A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast.

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.

Nup124p is a nuclear pore factor of Schizosaccharomyces pombe that is important for nuclear import and activity of retrotransposon Tf1.

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

Slk19p is a centromere protein that functions to stabilize mitotic spindles.

We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability. When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed. A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body. Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules.

Localization of the 26S proteasome during mitosis and meiosis in fission yeast.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the localization of this complex in the fission yeast, Schizosaccharomyces pombe. Immunofluorescence microscopy shows a striking localization pattern whereby the proteasome is found predominantly at the nuclear periphery, both in interphase and throughout mitosis. Electron microscopic analysis revealed a concentration of label near the inner side of the nuclear envelope. The localization of green fluorescent protein (GFP)-tagged 26S proteasomes was analyzed in live cells during mitosis and meiosis. Throughout mitosis the proteasome remained predominantly at the nuclear periphery. During meiosis the proteasome was found to undergo dramatic changes in its localization. Throughout the first meiotic division, the signal is more dispersed over the nucleus. During meiosis II, there was a dramatic re-localization, and the signal became restricted to the area between the separating DNA until the end of meiosis when the signal dispersed before returning to the nuclear periphery during spore formation. These findings strongly imply that the nuclear periphery is a major site of protein degradation in fission yeast both in interphase and throughout mitosis. Furthermore they raise interesting questions as to the spatial organization of protein degradation during meiosis.

The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control.

During fission yeast mitosis, the duplicated spindle pole bodies (SPBs) nucleate microtubule arrays that interdigitate to form the mitotic spindle. cut12.1 mutants form a monopolar mitotic spindle, chromosome segregation fails, and the mutant undergoes a lethal cytokinesis. The cut12(+) gene encodes a novel 62-kD protein with two predicted coiled coil regions, and one consensus phosphorylation site for p34(cdc2) and two for MAP kinase. Cut12 is localized to the SPB throughout the cell cycle, predominantly around the inner face of the interphase SPB, adjacent to the nucleus. cut12(+) is allelic to stf1(+); stf1.1 is a gain-of-function mutation bypassing the requirement for the Cdc25 tyrosine phosphatase, which normally dephosphorylates and activates the p34(cdc2)/cyclin B kinase to promote the onset of mitosis. Expressing a cut12(+) cDNA carrying the stf1.1 mutation also suppressed cdc25.22. The spindle defect in cut12.1 is exacerbated by the cdc25.22 mutation, and stf1.1 cells formed defective spindles in a cdc25.22 background at high temperatures. We propose that Cut12 may be a regulator or substrate of the p34(cdc2) mitotic kinase.

The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin.

The gene encoding the actin-related protein Arp3 was first identified in the fission yeast Schizosaccharomyces pombe and is a member of an evolutionarily conserved family of actin-related proteins. Here we present several key findings that define an essential role for Arp3p in the functioning of the cortical actin cytoskeleton. First, mutants in arp3 interact specifically with profilin and actin mutants. Second, Arp3 localizes to cortical actin patches which are required for polarized cell growth. Third, the arp3 gene is required for the reorganization of the actin cytoskeleton during the cell cycle. Finally, the Arp3 protein is present in a large protein complex. We believe that this complex may mediate the cortical functions of profilin at actin patches in S. pombe.

A mutation in the RCC1-related protein pim1 results in nuclear envelope fragmentation in fission yeast.

Members of the RCC1 protein family are chromatin-associated guanine nucleotide exchange factors that have been implicated in diverse cellular processes in various organisms, yet no consensus has been reached as to their primary biological role. The fission yeast Schizosaccharomyces pombe, a single-celled eukaryote, provides an in vivo system in which to study the RCC1/Ran switch by using a temperature-sensitive mutant in the RCC1-related protein pim1. Mitotic entry in the pim1-d1ts mutant is normal, but mitotic exit leads to the accumulation of cells arrested with a medial septum and condensed chromosomes. Although the yeast nuclear envelope normally remains intact throughout the cell cycle, we found a striking fragmentation of the nuclear envelope in the pim1-d1ts mutant following mitosis. This resulted in chromatin that was no longer compartmentalized and an accumulation of pore-containing membranes in the cytoplasm. The development of this terminal phenotype was dependent on the passage of cells through mitosis and was coincident with the loss of viability. We propose that pim1 is required for the reestablishment of nuclear structure following mitosis in fission yeast.

Improved preservation of ultrastructure in difficult-to-fix organisms by high pressure freezing and freeze substitution: I. Drosophila melanogaster and Strongylocentrotus purpuratus embryos.

In this study, we have applied the techniques of high pressure freezing and freeze substitution to embryonic cell types which are usually difficult to fix properly for electron microscopy. In both Drosophila and Strongylocentrotus purpuratus, we see improved preservation of both membrane systems and cytoskeleton when compared to published results on the same cells using conventional electron microscope (EM) fixation methods. Finally, we have seen that postembedding labelling of sections is possible even after light osmium fixation during freeze substitution.