表面扩散和酵母染色体免疫染色
Summary
A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.
Abstract
The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.
Introduction
在芽殖酵母提供了许多优势，为生物过程的分子机制，包括控制染色体功能的蛋白质的研究研究。虽然有出芽酵母基因，分子和生物化学研究的公知的优点，蛋白质在细胞核分布的细胞学研究是由它的小尺寸复杂。典型的酵母核的直径小于一微米，这是可见光的只有约5倍的分辨率极限。因此，对核蛋白质，可以从常规的免疫染色或通过使用荧光蛋白标记，如绿色荧光蛋白（GFP）来获得的分配信息的数量是有限的。一个有用的方法来表征蛋白质的亚核的分布是染色体的表面扩散。该方法包括去除细胞壁，扰乱细胞和核膜，和一个llowing核的不溶性内容沉降到显微镜载玻片的表面上。这些不溶性组分包括核基质和染色体。除了允许去除可溶性核内容，从而增强检测的染色质结合的蛋白质的能力，所述染色体扩频方法导致染色体大幅度减压，使得传播核具有的直径约3至5微米的（对于二倍体减数分裂核）和2至3微米为二倍体有丝分裂核。这种减压使检测核亚结构是相对困难或不可能解决在完整的细胞核。
一个明显的缺点，以染色体扩频这样的可能性，即在扩展过程可以部分或完全破坏感兴趣的结构。特别值得关注的是，一个特定的染色体结合蛋白可能会丢失作为扩展过程的结果。这种潜在的并发症寿LD解释数据时必须牢记这一点。的蛋白质的一个例子是于扩展过程敏感的β-微管蛋白。在某些情况下，主轴，其由主要的微管蛋白，被扩频3中保存。主轴的可视化往往是有益的舞台感兴趣的原子核。然而，可视化微管蛋白需要有高浓度的固定剂处理;主轴下述的标准条件下丢失。这个实施例说明，在分析以前未表征的蛋白质的分布时，它改变固定液以确定蛋白质如何敏感是这种变化的浓度是重要的。尽管关于扩频条件对染色体结构的影响的关注，扩展方法的实用性和功率已被证明在许多情况下，具有在有丝分裂的表征，特别是减数分裂细胞4-7广泛的用途。
TWø扩频方法已被广泛使用。第一这些方法，通过修整和吉鲁8研制，避免了使用洗涤剂的，并且可以得到显示时染色的DNA特异性染料DAPI具有相对保存良好染色体形态传播的制剂。然而，这种方法是比较困难的完善和传播核的质量显着地发生变化，当一个滑动件的一个区域被与其他区域相比。这个问题变得更复杂，涉及很多成像细胞核未选定从一个幻灯片，以避免数据采集偏见定量方法。第二种染色体传播方法，通过Loidl和克莱因1发达，要平衡固定由多聚甲醛，用裂解和染色质解由洗涤剂溶液提升。当适当地进行，该方法提供了用更少的区域对区域的变化非常可重复的结果相比，德莱和吉鲁方法。本演讲将重点在modified版，因为它的可靠性和简易Loidl和克莱因的方法，。
染色体扩展并不复杂或费时;高达100幻灯片可以用于免疫染色在一天制备。此外，传播制剂可贮存在冷冻多年之前免疫染色，因此实验室可以开发冷冻染色体差，可以当新的生物问题出现的或新的染色试剂变得可用可使用的存储库。
染色体扩频方法是最常用的与免疫染色和宽场荧光显微镜组合，但是也可能制备载玻片超分辨率光学显微镜方法如受激发射损耗（STED）显微镜。
Protocol
Representative Results
Discussion
The appearance of spread nuclei critically depends upon the balance between fixation and lysis/detergent treatment. As discussed above, the preservation of varying cellular structures requires the use of different PFA concentrations. For most proteins, 3% PFA is optimal. However, preservation of spindles requires use of 4% PFA. Even with a single set of reagents, the timing of lysis relative to fixation can also affect the quality of spread nuclei. For the most consistent results, the time of lysis should be normalize…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grant GM50936 to DKB.
Materials
| Zymolyase | US Biological | Z1004 | Prepare 20 mg/mL solution in 50 mM Tris pH 7.5 supplemented with 2% glucose. Prepare fresh each experiment and store at 4°C until ready for use. |
| Lipsol | L.I.P. Ltd | no longer commercially available | Prepare 1% (v/v) solution in water. Store on ice. |
| NP-40 | USB | 19628 | Prepare 1% (v/v) solution in water. Store on ice. |
| Tween 20 | Sigma | P2287 | |
| Slides | Corning | 2948-75×25 | |
| Standard coverslip | Fisher | 12-544-E or 12-540-B | |
| High resolution coverslips | Fisher | 12-542-B | |
| Photo-Flo 200 solution | Kodak | P-7417 | Prepare 0.2% (v/v) solution in water. |
| TBS | 137 mM NaCl, 2.7 mM KCl, 24.7 mM Tris, pH 8 | ||
| BSA | Sigma | A2153 | Prepare a 1% (w/v) solution in TBS. Store at 4°C for up to a month. |
| Primary antibody | |||
| Alexa Fluor 488 Donkey Anti-Rabbit | Invitrogen | A-21206 | |
| IgG (H+L) Antibody | |||
| Vectashield mounting media with DAPI | Vector Laboratories |
H-1200 | |
| ProLong Gold | Invitrogen | P36930 | |
| Plastic slide box | Fisher | 03-448-1 | Store slides containing dried spreads in slots at -20°C. Also, use as a wet chamber. |
| Cardboard slide box | Fisher | 12-587-10 | Use to conveniently transport stained/sealed slides or store at 4°C. |
| Coplin jar | Fisher | 08-816 | Use as a wash basin for slides. |
References
- Loidl, J., Nairz, K., Klein, F. Meiotic chromosome synapsis in a haploid yeast. Chromosoma. 100 (4), 221-228 (1991).
- Loidl, J., Klein, F., Engebrecht, J. Genetic and morphological approaches for the analysis of meiotic chromosomes in yeast. Methods in cell biology. 53, 257-285 (1998).
- Lydall, D., Nikolsky, Y., Bishop, D. K., Weinert, T. A meiotic recombination checkpoint controlled by mitotic checkpoint genes. Nature. 383 (6603), 840-843 (1996).
- Bishop, D. K. RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis. Cell. 79, 1081-1092 (1994).
- Rabitsch, K. P., Galova, M., Schleiffer, A., Buonomo, S. B., Nasmyth, K. Functional genomics identifies Monopolin: a kinetochore protein required for segregation of homologs during meiosis I. Cell. 103 (7), 1155-1168 (2000).
- Gasior, S. L., Olivares, H., Ear, U., Hari, D. M., Weichselbaum, R., Bishop, D. K. Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis. PNAS. 98 (15), 8411-8418 (2001).
- Biggins, S., Murray, A. W. The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint. Genes & Development. 15 (23), 3118-3129 (2001).
- Dresser, M. E., Giroux, C. N. Meiotic chromosome behavior in spread preparations of yeast. The Journal of Cell Biology. , (1988).
- Lao, J. P., Cloud, V., et al. Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation. PLOS Genetics. 9 (12), e1003978-e1003978 (2013).
- Vonesch, C., Unser, M. A fast thresholded landweber algorithm for wavelet-regularized multidimensional deconvolution. IEEE transactions on image processing : a publication of the. IEEE Signal Processing Society. 17 (4), 539-549 (2008).
