Stephen M Murray

Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast.

By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.

The Mitotic Exit Network and Cdc14 phosphatase initiate cytokinesis by counteracting CDK phosphorylations and blocking polarised growth.

Polarisation of the actin cytoskeleton must cease during cytokinesis, to support efficient assembly and contraction of the actomyosin ring at the site of cell division, but the underlying mechanisms are still understood poorly in most species. In budding yeast, the Mitotic Exit Network (MEN) releases Cdc14 phosphatase from the nucleolus during anaphase, leading to the inactivation of mitotic forms of cyclin‐dependent kinase (CDK) and the onset of septation, before G1‐CDK can be reactivated and drive re‐polarisation of the actin cytoskeleton to a new bud. Here, we show that premature inactivation of mitotic CDK, before release of Cdc14, allows G1‐CDK to divert the actin cytoskeleton away from the actomyosin ring to a new site of polarised growth, thereby delaying progression through cytokinesis. Our data indicate that cells normally avoid this problem via the MEN‐dependent release of Cdc14, which counteracts all classes of CDK‐mediated phosphorylations during cytokinesis and blocks polarised growth. The dephosphorylation of CDK targets is therefore central to the mechanism by which the MEN and Cdc14 initiate cytokinesis and block polarised growth during late mitosis.

Visualization of the nucleus and nuclear envelope in situ by SEM in tissue culture cells

Our previous work characterizing the biogenesis and structural integrity of the nuclear envelope and nuclear pore complexes (NPCs) has been based on amphibian material but has recently progressed into the analysis of tissue-culture cells. This protocol describes methods for the high resolution visualization, by field-emission scanning electron microscopy (FESEM), of the nucleus and associated structures in tissue culture cells. Imaging by fluorescence light microscopy shows general nuclear and NPC information at a resolution of approximately 200 nm, in contrast to the 3–5 nm resolution provided by FESEM or transmission electron microscopy (TEM), which generates detail at the macromolecular level. The protocols described here are applicable to all tissue culture cell lines tested to date (HeLa, A6, DLD, XTC and NIH 3T3). The processed cells can be stored long term under vacuum. The protocol can be completed in 5 d, including 3 d for cell growth, 1 d for processing and 1 d for imaging.

High pressure freezing and freeze substitution of Schizosaccharomyces pombe and Saccharomyces cerevisiae for TEM.

The use of standard room temperature chemical fixation protocols for the ultrastructural preservation of yeast and subsequent observation under the electron microscope is fraught with difficulties. Many protocols require the use of enzymatic digestion of the cell wall in order to facilitate the entry of fixatives into the cell interior. Others rely on the use of permanganate-based fixative solutions, which whilst enabling overall preservation of the cell, does require multiple centrifugation, washing, and resuspension steps. This often results in the significant loss of sample volume whilst the use of permanganate can cause extraction of cytoplasmic components. The use of low temperature techniques and in particular high pressure freezing (HPF) and freeze substitution (FS) overcomes many of these problems. With the recent advances in cryotechnologies and in particular the development of commercially available equipment such as the high pressure freezer, the level of ultrastructural preservation attainable in electron microscopy has increased markedly. It is now possible to capture dynamic time sensitive events and to place them in their ultrastructural context with a level of resolution which at the present time can only be achieved with electron microscopy.

A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3–5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.

Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for Western blotting and scanning electron microscopy (SEM).

This protocol details methods for the generation of cell-free extracts and DNA templates from the eggs and sperm chromatin, respectively, of the clawed toad Xenopus laevis. We have used this system with scanning electron microscopy (SEM), as detailed herein, to analyze the biochemical requirements and structural pathways for the biogenesis of eukaryotic nuclear envelopes (NEs) and nuclear pore complexes (NPCs). This protocol requires access to female frogs, which are induced to lay eggs, and a male frog, which is killed for preparation of the sperm chromatin. Egg extracts should be prepared in 1 d and can be stored for many months at −80 °C. Demembranated sperm chromatin should take only approximately 2–3 h to prepare and can be stored at −80 °C almost indefinitely. The time required for assembly of structurally and functionally competent nuclei in vitro depends largely on the quality of the cell-free extracts and, therefore, must be determined for each extract preparation.

Scanning electron microscopy of nuclear structure.

Accessing internal structure and retaining relative three dimensional (3D) organization within the nucleus has always proved difficult in the electron microscope. This is due to the overall size and largely fibrous nature of the contents, making large scale 3D reconstructions difficult from thin sections using transmission electron microscopy. This chapter brings together a number of methods developed for visualization of nuclear structure by scanning electron microscopy (SEM). These methods utilize the easily accessed high resolution available in field emission instruments. Surface imaging has proved particularly useful to date in studies of the nuclear envelope and pore complexes, and has also shown promise for internal nuclear organization, including the dynamic and radical reorganization of structure during cell division. Consequently, surface imaging in the SEM has the potential to make a significant contribution to our understanding of nuclear structure.

A Protocol for Isolation and Visualization of Yeast Nuclei by Scanning Electron Microscopy

This article describes a protocol that details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immunogold labelling of proteins, and visualization by Field Emission Scanning Electron Microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high resolution microscopy. The nuclear isolation step is performed by enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of nuclei by centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs), and associated cytoskeletal structures. Samples, once processed for FESEM, can be stored under vacuum for weeks, allowing considerable time for image acquisition.

IP3R1 - Assessing Map Interpretability at Near Atomic Resolution

Once resolutions beyond ~6 Å have been achieved in single particle reconstruction it becomes possible to begin interpreting maps in terms of the protein sequence. At the low resolution extreme, C-alpha tracing and homology modeling can be applied. As resolution increases towards 4 Å, beta strand separation becomes clear, and larger sidechain densities begin to appear. However, simply citing the resolution of a map, regardless of criteria, does not provide sufficient information to assess the appropriate level of interpretability. It is always possible to filter maps such that, for example, sidechain densities appear, even when such densities are likely due primarily to noise.