Kenneth E. Sawin

Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts

Using Xenopus egg extracts arrested in interphase or mitosis, we directly observed differences in microtubule dynamics at different stages of the cell cycle. Interphase extracts were prepared from eggs in the first interphase after meiosis. Mitotic extracts were prepared by addition of purified cyclin to interphase extracts. Microtubules were nucleated by the addition of centrosomes and visualized by fluorescence video-microscopy in extracts to which rhodamine-labeled tubulin had been added. We found a striking difference in microtubule dynamics in mitotic versus interphase extracts. Quantitative analysis revealed that the rates of polymerization and depolymerization are similar in interphase and mitosis and that within the spatial and temporal resolution of our experiments the difference in dynamics is due almost entirely to an increase in the frequency of transition from growing to shrinking (catastrophe frequency) in the mitotic extracts.

Microtubule Nucleation at Non-Spindle Pole Body Microtubule-Organizing Centers Requires Fission Yeast Centrosomin-Related Protein mod20p

BACKGROUND Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. RESULTS From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. CONCLUSIONS Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.

Caged fluorescent probes

Publisher Summary This chapter discusses fluorescence photoactivation as an alternative technology for probing cytoskeleton dynamics. In this approach, the protein is tagged with a caged fluorochrome. This probe molecule is nonfluorescent until illuminated with a brief pulse of ultraviolet light. Such illumination leads to photolysis of the caging groups and generation of a fluorescent species. In principle, fluorescence photoactivation can avoid the problem of generation of local oxidative damage inherent to photobleaching and can also produce a more favorable signal-to-noise ratio for imaging. Photoactivation can also produce toxic by-products in the form of the nitrosoaldehyde or nitrosoketone side products from photolysis. The chapter discusses the progress in caged fluorescent probes. The first caged fluorescent probe that was used for a biological experiment was a fluorescein derivative, C2CF-sulfo-N-hydroxysuccinimide. This probe, attached to tubulin, led to the discovery of poleward flux in mitotic spindles. C2CF is highly hydrophobic, and most proteins other than tubulin tend to aggregate if they are labeled with it.

Fission yeast mod5p regulates polarized growth through anchoring of tea1p at cell tips

Microtubules have a central role in eukaryotic cell polarity, in part through interactions between microtubule end-binding proteins and the cell cortex. In the fission yeast Schizosaccharomyces pombe, microtubules and the polarity modulator tea1p maintain cylindrical cell shape and strictly antipodal cell growth. The tea1p protein is transported to cell tips by association with growing microtubule plus ends; once at cell tips, tea1p releases from microtubule ends and associates with the cell cortex, where it coordinates polarized growth. Here we describe a cortical protein, mod5p, that regulates the dynamic behaviour of tea1p. In mod5Δ cells, tea1p is efficiently transported on microtubules to cell tips but fails to anchor properly at the cortex and thus fails to accumulate to normal levels. mod5p contains a signal for carboxy-terminal prenylation and in wild-type cells is associated with the plasma membrane at cell tips. However, in tea1Δ cells, although mod5p remains localized to the plasma membrane, mod5p is no longer restricted to the cell tips. We propose that tea1p and mod5p act in a positive-feedback loop in the microtubule-mediated regulation of cell polarity.

Cytoplasmic microtubule organization in fission yeast.

During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These may serve as a model for understanding the different modes of microtubule organization that are often characteristic of differentiated higher eukaryotic cells. In the last few years, considerable progress has been made in our understanding of the organization and behaviour of fission yeast cytoplasmic microtubules, not only in the identification of the genes and proteins involved but also in the physiological analysis of function using fluorescently‐tagged proteins in vivo. In this review we discuss the state of our knowledge in three areas: microtubule nucleation, regulation of microtubule dynamics and the organization and polarity of microtubule bundles. Advances in these areas provide a solid framework for a more detailed understanding of cytoplasmic microtubule organization. Copyright

Multistep and multimode cortical anchoring of tea1p at cell tips in fission yeast

The fission yeast cell‐polarity regulator tea1p is targeted to cell tips by association with growing microtubule ends. Tea1p is subsequently anchored at the cell cortex at cell tips via an unknown mechanism that requires both the tea1p carboxy‐terminus and the membrane protein mod5p. Here, we show that a tea1p‐related protein, tea3p, binds independently to both mod5p and tea1p, and that tea1p and mod5p can also interact directly, independent of tea3p. Despite their related structures, different regions of tea1p and tea3p are required for their respective interactions with an essential central region of mod5p. We demonstrate that tea3p is required for proper cortical localization of tea1p, specifically at nongrowing cell tips, and that tea1p and mod5p are independently required for tea3p localization. Further, we find that tea3p fused to GFP or mCherry is cotransported with tea1p by microtubules to cell tips, but this occurs only in the absence of mod5p. These results suggest that independent protein–protein interactions among tea1p, tea3p and mod5p collectively contribute to tea1p anchoring at cell tips via a multistep and multimode mechanism.

Role of microtubules and tea1p in establishment and maintenance of fission yeast cell polarity

Microtubules and the protein tea1p have important roles in regulating cell polarity in the fission yeast Schizosaccharomyces pombe. Here, using combinations of drugs, environmental perturbations and genetic mutants, we demonstrate that once a cell polarity axis is established, microtubules have at best a minor role in maintaining the cortical actin cytoskeleton and the rate and direction of cell growth. In addition, we find that after perturbations that disrupt cell polarity and the cortical actin cytoskeleton, microtubules are not required for re-establishment of polarity per se. However, after such perturbations, the distribution of cytoplasmic microtubules plays an important role in dictating the position of sites of polarity re-establishment. Furthermore, this influence of microtubule distribution on site selection during polarity re-establishment requires the presence of tea1p, suggesting that tea1p is crucial for coupling microtubule distribution to the regulation of cell polarity. Our results suggest a model in which, at the cellular level, two distinct and separable mechanisms contribute to how tea1p regulates site selection during polarity re-establishment. First, tea1p remaining at cell tips after cortical depolarization can serve as a cortical landmark for microtubule-independent site selection; second, tea1p newly targeted to the cell cortex by association with microtubules can promote the formation of polarity axes de novo.

Motor proteins in cell division

The movements of eukaryotic cell division depend upon the conversion of chemical energy into mechanical work, which in turn involves the actions of motor proteins, molecular transducers that generate force and motion relative cytoskeletal elements. In animal cells, microtubule-based motor proteins of the mitotic apparatus are involved in segregating chromosomes and perhaps in organizing the mitotic apparatus itself, while microfilament-based motors in the contractile ring generate the forces that separate daughter cells during cytokinesis. This review outlines recent advances in our understanding of the roles of molecular motors in mitosis and cytokinesis.

Photoactivation of green fluorescent protein.

Considerable insight into many cell biological processes can be obtained by following the turnover of individual protein species in time and space. In living cells, this has been achieved by following fluorescence recovery after local microbeam photobleaching of microinjected fluorescently labelled protein (see, for example, [1xSpindle microtubule dynamics in sea urchin embryos: analysis using fluorescence-labelled tubulin and measurements using fluorescence redistribution after laser photobleaching. Salmon, ED, Leslie, RJ, Saxton, WM, Karow, ML, and Mclntosh, JR. J Cell Biol. 1984; 99: 2165–2174Crossref | PubMed | Scopus (108)See all References[1]) as well as by photoactivation of ‘caged’ fluorescently labelled proteins [[2]xPolewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. Mitchison, TJ. J Cell Biol. 1989; 109: 637–652Crossref | PubMedSee all References, [3]xPhotoactivation of fluorescence as a probe for cytoskeletal dynamics in mitosis and cell motility. Sawin, KE, Theriot, JA, and Mitchison, TJ. See all References].The gene encoding the naturally occurring green fluorescent protein (GFP) of the cnidarian Aequorea victoria[4xPrimary structure of the Aequorea victoria green-fluorescent protein. Prasher, DC, Eckenrode, VK, Ward, WW, Prendergast, FG, and Cormier, MJ. Gene. 1992; 111: 229–233Crossref | PubMed | Scopus (1425)See all References[4] has proved invaluable as an in vivo fluorescence tag for the subcellular localization of proteins and/or as a reporter of specific promoter and/or enhancer activities in a multitude of organisms [5xGreen fluorescent protein as a marker for gene expression. Chalfie, M, Tu, Y, Euskirchen, G, Ward, WW, and Prasher, DC. Science. 1994; 263: 802–805Crossref | PubMedSee all References[5]. Recently, GFP has been used to study protein dynamics in cells using photobleaching [[6]xDiffusional mobility of Golgi proteins in membranes of living cells. Cole, NB, Smith, CL, Sciaky, L, Terasaki, M, Edidin, M, and Lippincott-Schwartz, J. Science. 1996; 273: 797–800Crossref | PubMedSee all References, [7]xCalcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores. Subramanian, K and Meyer, T. Cell. 1997; 89: 963–971Abstract | Full Text | Full Text PDF | PubMedSee all References] and local fluorescence enhancement [8xSpatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Yokoe, H and Meyer, T. Nature Biotech. 1996; 14: 1252–1256CrossrefSee all References[8]. Here, we describe in vivo photoactivation of GFP to a novel red fluorescent form by illumination with blue light. We hope this method will be of use and interest to many investigators.Fission yeast (Schizosaccharomyces pombe) expressing the red-shifted variant GFPmut2 [9xFACS-optimized mutants of the green fluorescent protein (GFP). Cormack, BP, Valdivia, RH, and Falkow, S. Gene. 1996; 173: 33–38Crossref | PubMed | Scopus (2071)See all References[9] in the multicopy plasmid pSGA [10xIdentification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein. Sawin, KE and Nurse, P. Proc Natl Acad Sci USA. 1996; 94: 15146–15151Crossref | Scopus (70)See all References[10] were immobilized at low density on a thin pad of EMM2 minimal medium containing 2% agarose and sealed under a coverslip with paraffin wax. Under these conditions cells can divide for several generations at nearly normal rates. After 2–3 cell divisions we illuminated microcolonies of cells with blue light (fluorescein filter set; 460–500 nm excitation), observing red fluorescence (Texas Red filter set; 540–580 nm excitation, 610–680 nm emission) before and after blue illumination (Figure 1Figure 1).Figure 1Photoactivation of GFP to a red-fluorescent protein on exposure to blue light. (a) DIC image of a microcolony of cells growing on an agarose pad in a sealed chamber. (b) Taxes Red image of the same microcolony before photoactivation. (c) Taxes Red image (same exposure time) after 2 sec photoactivation, using blue light from the fluorescein filter set. (d) Fluorescein image, at the end of the experiment. The bottom four cells are expressing less protein, probably because of plasmid mis-segregation during the first division. Note that photoactivated red fluorescence is proportional to green fluorescence, and that faint background fluorescence in the Texas Red channel (from out-of-focus debris) does not appear in the fluorescein channel. Bar is 10 μm. Images were collected on a Power Macintosh 8500 computer using a Hamamatsu C5985 chilled video-rate CCD mounted on a Zeiss Axiomat microscope (40×/0.7 Plan-Neofluar objective) equipped with High-Q filter sets (Chroma Technology, Brattleboro, Vermont). Exposure times were 0.04 sec for DIC, 2 sec for both Texas Red images, and 0.2 sec for the fluorescein image; no further image processing was applied.View Large Image | View Hi-Res Image | Download PowerPoint SlideWe found that relatively short exposures to blue light (1–5 sec) are sufficient to generate a significant photoconversion of GFPmut2 to a stable red fluorescent form, and that the amount of red fluorescence produced is proportional to the total green fluorescence. When viewed with a Cy5 filter set (590–650 nm excitation, 670–730 nm emission), the signal was considerably more faint; with a tetramethyl rhodamine filter set (510–555 nm excitation, 570–640 nm emission), cells were already visible before photoactivation, but became brighter with increasing exposure. We have observed photoactivation with several different GFP fusion proteins, located in the cytoplasm and in the nucleus, and associated with the plasma membrane.Although attempts to photoactivate cells taken directly from exponentially growing shaken cultures were unsuccessful, cells taken from a pellet after a brief (5–10 min) centrifugation were easily photoactivated, whether in minimal medium or in a phosphate-buffered saline solution. Photoactivatability was immediately lost on vigorous shaking of the centrifuged cells. Isolation from the atmosphere thus seems to be an important factor in photoactivation, consistent with additional observations that cells at the periphery of an unsealed chamber are poorly activated, whereas those in the center activate readily. One possible reason for this could be that some amount of oxygen depletion from the system may be required for efficient photoactivation; fission yeast grow completely normally under non-aerated, and thus near-anaerobic, conditions [11xPhysiological and cytological methods for Schizosaccharomyces pombe. Mitchison, JM. See all References[11].Although the mechanisms underlying photoactivation of GFP are not yet clear, photoactivation does not seem to be specific to GFPmut2, as we have observed similar effects with both wild-type GFP and the S65T mutant [12xImproved green fluorescence. Heim, R, Cubitt, AB, and Tsien, RY. Nature. 1995; 373: 663–664Crossref | PubMedSee all References[12]— although wild-type GFP was photoactivated with ultraviolet light (340–380 nm) more efficiently than with blue light — in all cases only under the conditions described above.Single-celled eukaryotes such as yeast have been for the most part ignored in studies of intracellular protein dynamics because they are recalcitrant to microinjection. Irrespective of the exact mechanisms involved in photoactivation, our observations demonstrate that photoactivating GFP into a red fluorescent protein will be a useful tool for cell biology, and we hope that these methods will be easily adaptable to other systems, including mammalian cells. Photoactivation of GFP fusion proteins would circumvent the need to microinject cells of all types and, in combination with local microbeaming of subcellular regions, should allow for interesting new insights into the dynamic behavior of protein assemblies within living cells.

A Genetic Engineering Solution to the “Arginine Conversion Problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.