我们要感谢秀丽遗传学中心的菌株, 自然科学和工程研究理事会 (NSERC) 和加拿大创新基金会 (CFI) 供资。我们要感谢关丽珍博士 (细胞系统和解剖学部, UT 健康圣安东尼奥) 允许不受限制地使用她的实验室设施为所有C. 线虫实验和 Exing 博士 (副主任, 光学成像设备, UT 健康圣安东尼奥) 为协助与共焦显微术。此外, 我们亦多谢何议员提供支持和鼓励, 以协助拍摄影片。

1. 染色和图像肠道溶酶体
<ol><li>OP50 种子线虫生长培养基 (NGM) 板
	<ol>
<li>根据建议的协议19准备 NGM 板, 并允许在室温下将密封板干燥2天。</li>
		<li>将 OP50 细菌接种到无菌 Luria 汤 (磅) 汤中, 在37摄氏度或36小时的水浴中生长, 或直到外径介于0.2 和0.4 之间。避免使用细菌培养 > 1 或 od < 0.2。</li>
		<li>一旦 NGM 板干燥, 将一滴 (~ 30 µL) 的 OP50 接种到板的中心, 然后把下降到一个补丁大致是2厘米 x 2 厘米的大小。 注: 任何剩余的 OP50 接种可以储存在4摄氏度, 长达一个月。</li>
		<li>在接种干燥后反转 OP50 板 (大约10或15分钟), 然后将37摄氏度的板材孵化为36小时。</li>
		<li>36小时后, 从孵化箱中取出盘子, 储存在4摄氏度, 直到以后使用。 注: 板材应在4摄氏度的1月内良好。</li>
	</ol></li>
	<li>补充 cDCFDA 到 OP50
	<ol>
<li>在 OP50 板上补充 cDCFDA, 在二甲基亚砜 (亚砜) 上制备10毫米 cDCFDA 的工作库存。保持 cDCFDA 解决方案免受光照和存储在-20 摄氏度后使用。</li>
		<li>轻轻地将100µL 10 毫米 cDCFDA 溶液放到2厘米 x 2 厘米 OP50 贴片的表面, 这样整个贴片都被 cDCFDA 均匀覆盖。 注意: 这是非常重要的, 整个补丁的 OP50 覆盖 cDCFDA。如果需要, 使用多达150µL 的 cDCFDA 为每个板块。cDCFDA 解决方案也必须不超出 OP50 补丁的边界范围, 因为从 OP50 修补程序流出的任何 cDCFDA 不会被纳入食物中, 而且会导致染色强度降低。</li>
		<li>将 OP50 板放在工作台顶部的一个黑盒子里, 直到所有的 cDCFDA 溶液都被吸收到细菌斑块 (通常约25到30分钟)。</li>
	</ol></li>
	<li>使用 cDCFDA 染色蠕虫
	<ol>
<li>一旦 cDCFDA 完全渗透到 OP50 补丁, 每板放置不超过20蠕虫和孵化的板块反转在20°c 至少14小时。 注意: 建议将 cDCFDA 板放在不透明的盒子中, 以尽量减少光的照射。</li>
		<li>为了控制实验之间的摄入差异和规范 cDCFDA 信号, (推荐) 与 3-{2-[(1 H) 的 co 染色, 1 ' H-22 '-bipyrrol 5-基 kappaN (1)) methylidene]-2 H-咯-5-基 kappaN} N [2-(二甲基氨基) 乙] propanamidato) (difluoro) 硼 (2.5 µL 的新鲜制成的1毫米溶液), acidotropic 弱胺染料, 其荧光主要是非变异的酸性 pH 谱 (参见材料表的常用试剂名称)。 注意: 不要将蠕虫染色少于14小时, 因为这可能会提供不充分的 cDCFDA 染色强度, 并对溶酶体 pH 值作假解释。</li>
	</ol></li>
	<li>准备用于显微镜的幻灯片
	<ol>
<li>将两条带标签的磁带贴在平滑的平坦表面上, 如镜像 (图 1), 其间隔约为3英寸。</li>
		<li>将镜面表面涂上防水喷雾剂, 并擦拭多余液体。</li>
		<li>熔融2% 琼脂糖 (溶解在蒸馏 H2O), 直到完全液化, 然后在两条胶带之间放置一个50µL 滴琼脂糖, 用显微镜滑动迅速覆盖下降, 这样, 幻灯片垂直于带磁带。在琼脂糖凝固后, 在滑块下形成一个圆形垫, 翻转滑块, 将10到15µL 的5mM 钠叠氮化物 (NaN3) 添加到琼脂糖垫上。 注意: NaN3是一种细胞色素 C 抑制剂, 当用于低浓度时, 可逆 anesthetizes 蠕虫, 以便它们在成像过程中不会移动。</li>
		<li>从 OP50 板中提取蠕虫补充 cDCFDA, 并将其转移到一个干净的 NGM 板没有任何 OP50。允许蠕虫移动大约几秒钟, 允许大部分 OP50 从蠕虫表面移除, 然后将蠕虫转移到含有5毫米叠氮化钠的琼脂糖垫上。</li>
		<li>立即用盖子滑盖琼脂糖垫。一定要轻轻地放置在不施加太多的压力, 因为这可能导致蠕虫爆裂的封面滑动。 注: OP50 细菌辅以 cDCFDA 荧光显微镜下可视化, 因此, 重要的是要尽可能最好地清洁蠕虫之前, 把它们放在含有叠氮化钠的琼脂糖垫。如果准备6多株线虫(包括 N2、野生型控制), 最好在6批次中制备样品, 以确保蠕虫不会在琼脂糖垫上干燥。在某些菌株中, 由于来自盖板的压力, 外阴在延长的时间内开始破裂, 因此有必要进行相应的计划。</li>
	</ol></li>
	<li>共焦显微术 注: 某些显微镜具有内置功能, 可自动增加荧光强度, 以补偿可变水平的荧光。这种显微镜对 cDCFDA 染色的蠕虫可能无法正常工作, 因为它们本身就会增加样品的荧光强度。<ol>
<li>在标准共焦显微镜上进行成像, 使用激发/发射波长设置为 cDCFDA 488/530 nm。采用单平面图像 (不是 Z 栈) 的肠道溶酶体, 只使用集中 (最大信号强度) 的溶酶体进行强度量化。 注: 也许是由于染料的可用性不同, 肠细胞溶酶体直接后咽后保持 cDCFDA 染色强度, 无论基因型或年龄, 因此应注意避免这些细胞的成像溶酶体。</li>
		<li>图像的幻灯片包含2天老野生类型 N2 蠕虫首先。在这样做的同时, 调整共焦成像参数, 如激光功率, 针孔, 光圈, 以确保 cDCFDA 染色强度不饱和。 注意: 设置了这些设置后, 不要在当天的整个映像期间更改它们。</li>
		<li>使用60X 放大透镜捕获单个小肠平面的 1024 x 1024 像素图像 (每个蠕虫的3到4个图像)。 注: 蠕虫的 cDCFDA 发射光谱将产生一个单一的突出峰值在520毫微米与它报告的荧光光谱20, 提供一个特定的信号读数, 有别于肠道自发荧光 (图 3)。</li>
		<li>将原始共焦图像 (. lsm 文件) 导出为每个通道的. tiff 文件。</li>
		<li>接下来, 打开 ImageJ, 然后单击文件 |打开以打开要量化的图像文件。图像加载后, 使用 ImageJ 中的 "椭圆形状选择" 工具选择感兴趣的区域 (ROI)。</li>
		<li>此后, 单击分析 |测量以量化该 ROI 的荧光强度。选择第4-5 不同的聚焦溶酶每个图像和计算平均相对荧光强度每个区域的利益 (ROI)。</li>
		<li>对于每个图像, 测量溶酶体之间肠道区域的背景荧光强度。从溶酶体荧光强度值中减去背景荧光强度值。</li>
		<li>总计, 收集大约30到50个单独规范化的价值为 cDCFDA 强度 (6 到10动物) 为每株被测试。使用这些值绘制一个框和晶须图, 使用任何适当的统计软件。 注: 染色可能因温度、潜伏期和动物整体健康/年龄等因素的不同而有很大差异, 因此荧光强度比较仅在样品过程中与同一染色试验有关。因此, 在每个染色实验中处理控制是很重要的。使用任何适当的统计软件计算统计差异 (t测试)。</li>
		<li>图像的所有菌株从同一实验 (当天染色) 顺序, 注意不要改变任何成像参数。</li>
	</ol></li>
</ol>

<ol>
	<li>Erjavec, N., Larsson, L., Grantham, J., Nystrom, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+aging+and+failure+to+segregate+damaged+proteins+in+Sir2+mutants.">Accelerated aging and failure to segregate damaged proteins in Sir2 mutants.</a> Genes Dev. 21 (19), 2410-2421 (2007).</li><li>Madeo, F., Eisenberg, T., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+for+the+avoidance+of+neurodegeneration.">Autophagy for the avoidance of neurodegeneration.</a> Genes Dev. 23 (19), 2253-2259 (2009).</li><li>Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+intracellular+protein-degradation+pathways+in+neurodegeneration.">The roles of intracellular protein-degradation pathways in neurodegeneration.</a> Nature. 443 (7113), 780-786 (2006).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313 (5793), 1604-1610 (2006).</li><li>Cuervo, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+aging:+the+importance+of+maintaining+&amp;#34;clean&amp;#34;+cells.">Autophagy and aging: the importance of maintaining &amp;#34;clean&amp;#34; cells.</a> Autophagy. 1 (3), 131-140 (2005).</li><li>Harrington, A. J., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+model+system+for+identifying+effectors+of+alpha-synuclein+misfolding+and+dopaminergic+cell+death+associated+with+Parkinson's+disease.">Caenorhabditis elegans as a model system for identifying effectors of alpha-synuclein misfolding and dopaminergic cell death associated with Parkinson's disease.</a> Methods. 53 (3), 220-225 (2011).</li><li>Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+amyloid+formation,+and+neurodegeneration:+a+critical+role+for+molecular+chaperones?.">Protein misfolding, amyloid formation, and neurodegeneration: a critical role for molecular chaperones?.</a> Neuron. 35 (1), 9-12 (2002).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+decline+in+chaperone-mediated+autophagy.">Age-related decline in chaperone-mediated autophagy.</a> J Biol Chem. 275 (40), 31505-31513 (2000).</li><li>Tonoki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+evidence+linking+age-dependent+attenuation+of+the+26S+proteasome+with+the+aging+process.">Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process.</a> Mol Cell Biol. 29 (4), 1095-1106 (2009).</li><li>Squier, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+protein+aggregation+during+biological+aging.">Oxidative stress and protein aggregation during biological aging.</a> Exp Gerontol. 36 (9), 1539-1550 (2001).</li><li>Sarkar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+enhance+autophagy+and+reduce+toxicity+in+Huntington's+disease+models.">Small molecules enhance autophagy and reduce toxicity in Huntington's disease models.</a> Nat Chem Biol. 3 (6), 331-338 (2007).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Boya, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+function+and+dysfunction:+mechanism+and+disease.">Lysosomal function and dysfunction: mechanism and disease.</a> Antioxid Redox Signal. 17 (5), 766-774 (2012).</li><li>Kim, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-aging+treatments+slow+propagation+of+synucleinopathy+by+restoring+lysosomal+function.">Anti-aging treatments slow propagation of synucleinopathy by restoring lysosomal function.</a> Autophagy. 12 (10), 1849-1863 (2016).</li><li>Vila, M., Bove, J., Dehay, B., Rodriguez-Muela, N., Boya, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+membrane+permeabilization+in+Parkinson+disease.">Lysosomal membrane permeabilization in Parkinson disease.</a> Autophagy. 7 (1), 98-100 (2011).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+intracellular+proteolytic+systems+change+with+age?.">How do intracellular proteolytic systems change with age?.</a> Front Biosci. 3, d25-d43 (1998).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+lysosomes+get+old.">When lysosomes get old.</a> Exp Gerontol. 35 (2), 119-131 (2000).</li><li>Gachet, Y., Codlin, S., Hyams, J. S., Mole, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=btn1,+the+Schizosaccharomyces+pombe+homologue+of+the+human+Batten+disease+gene+CLN3,+regulates+vacuole+homeostasis.">btn1, the Schizosaccharomyces pombe homologue of the human Batten disease gene CLN3, regulates vacuole homeostasis.</a> J Cell Sci. 118 (Pt 23), 5525-5536 (2005).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Preston, R. A., Murphy, R. F., Jones, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+vacuolar+pH+in+yeast+and+identification+of+acidification-defective+mutants.">Assay of vacuolar pH in yeast and identification of acidification-defective mutants.</a> Proc Natl Acad Sci U S A. 86 (18), 7027-7031 (1989).</li><li>Pringle, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy+methods+for+yeast.">Fluorescence microscopy methods for yeast.</a> Methods Cell Biol. 31, 357-435 (1989).</li><li>Baxi, K., Ghavidel, A., Waddell, B., Harkness, T. A., de Carvalho, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Lysosomal+Function+by+the+DAF-16+Forkhead+Transcription+Factor+Couples+Reproduction+to+Aging+in+Caenorhabditis+elegans.">Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis elegans.</a> Genetics. 207 (1), 83-101 (2017).</li><li>Colacurcio, D. J., Nixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+lysosomal+acidification-The+emerging+role+of+v-ATPase+in+aging+and+neurodegenerative+disease.">Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease.</a> Ageing Res Rev. 32, 75-88 (2016).</li><li>Kang, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+screening+identifies+ATM+as+a+target+for+alleviating+senescence.">Chemical screening identifies ATM as a target for alleviating senescence.</a> Nat Chem Biol. 13 (6), 616-623 (2017).</li><li>Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R., Finkbeiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+formation+reduces+levels+of+mutant+huntingtin+and+the+risk+of+neuronal+death.">Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.</a> Nature. 431 (7010), 805-810 (2004).</li><li>Brunk, U. T., Terman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial-lysosomal+axis+theory+of+aging:+accumulation+of+damaged+mitochondria+as+a+result+of+imperfect+autophagocytosis.">The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.</a> Eur J Biochem. 269 (8), 1996-2002 (2002).</li><li>Gerland, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autolysosomes+accumulate+during+in+vitro+CD8++T-lymphocyte+aging+and+may+participate+in+induced+death+sensitization+of+senescent+cells.">Autolysosomes accumulate during in vitro CD8+ T-lymphocyte aging and may participate in induced death sensitization of senescent cells.</a> Exp Gerontol. 39 (5), 789-800 (2004).</li><li>Poon, H. F., Vaishnav, R. A., Getchell, T. V., Getchell, M. L., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+analysis+of+differential+protein+expression+and+oxidative+modification+of+specific+proteins+in+the+brains+of+old+mice.">Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice.</a> Neurobiol Aging. 27 (7), 1010-1019 (2006).</li><li>Yin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+basis+of+lipofuscin,+ceroid,+and+age+pigment-like+fluorophores.">Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores.</a> Free Radic Biol Med. 21 (6), 871-888 (1996).</li><li>Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reduction+in+intestinal+cell+pHi+due+to+loss+of+the+Caenorhabditis+elegans+Na+/H++exchanger+NHX-2+increases+life+span.">A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.</a> J Biol Chem. 278 (45), 44657-44666 (2003).</li><li>Chakraborty, K., Leung, K., Krishnan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+lumenal+chloride+in+the+lysosome+is+critical+for+lysosome+function.">High lumenal chloride in the lysosome is critical for lysosome function.</a> Elife. 6, (2017).</li></ol>

作者声明没有利益冲突。

各种细胞和分子事件导致衰老, 受生命史特征和遗传因素的影响。我们最近的研究22表明, 生殖周期通过调节溶酶体 pH 动态, 在控制躯体健康方面起着重要作用。我们表明, 溶酶介导的蛋白质水解是促进, 而动物积极繁殖的 v-atp 酶转录上调, 这反过来确保酸性溶酶。繁殖结束后, v-atp 酶表达下降, 溶酶体 alkalinize, 蛋白质聚集积累在这些细胞。
损害溶酶体酸化的?...

蛋白质团聚体的出现被广泛认为是真核细胞老化的标志1,2,3, 和其形成是细胞衰老的原则驱动因素4,5,6,7. 越来越多的证据表明, 随着细胞老化, 蛋白质分解代谢被削弱, 导致蛋白质聚集的增加。衰老细胞蛋白质水解的崩溃涉及自噬8的损伤以及蛋白酶介导的蛋白质降解9。最后, 在旧细胞中增加了不可逆转的蛋白质氧化, 进一步损害了蛋白质的分解代谢10。
自噬最初被认为是一个非选择性的过程, 以大量降解受损的蛋白质, 但最近的研究表明, 自噬是高度选择性的分解代谢的蛋白质聚集和功能失调的细胞器, 不可通过其他蛋白质清除机制11进行降解。在自噬过程中, 受损和聚集的蛋白质被隔离成一个叫做 autophagosome 的双膜囊泡。这个 autophagosome 然后与称为溶酶体的酸性细胞器融合, 导致 autophagosome 货物的降解12。溶酶体代表自噬性通路的终点, 参与不同的细胞过程, 如膜修复、转录控制和养分传感;突出它们在细胞稳态中的集中作用 (在 ref 13 中复习)。一些研究表明, 年龄依赖性的溶酶体功能下降与各种神经退行性疾病的相关性为13。一贯地, 在旧的细胞中恢复溶酶函数可以延缓衰老相关表型的出现,14,15。对 intralumen 环境组成的研究表明, 早期细胞溶酶酶功能的崩溃不是由于溶酶酶蛋白酶16的产生而减少的。另外, 有人建议, intralysosomal 酸度的损失, 它的酶活性的一个关键要求, 可能是在溶酶介导的蛋白质降解17的下降。为了能够探索这一假说, 必须开发试剂和协议, 以一种可复制和一致的方式来探测活细胞中溶酶体 pH 值的动态变化。
线虫的肠道是蠕虫的主要代谢组织, 是系统性稳态和寿命的重要调节因子。我们已经开发了测定蠕虫肠道溶酶体的酸性变化的检测方法, 以确定溶酶介导的蛋白质水解如何导致衰老。虽然以前在线虫中使用了 pH 敏感的显影标记肠道溶酶体, 但还没有努力建立一个成功的协议, 可以检测溶酶体 pH体内的小增加18.在这里, 我们提供了一种协议, 可用于检测C. 线虫肠道细胞中溶酶体酸度的损失, 使用一种简单而方便的喂养协议, 将 pH 敏感的荧光 (cDCFDA) 纳入 OP50 食品中。

<table border="0" cellpadding="0" cellspacing="0" width="1166">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:475px;">OP50 (E. coli)</td>
			<td style="width:188px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td style="width:385px;">Order online at https://cgc.umn.edu/strain/OP50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">5(6)-carboxy-2&#8217;,7&#8217;-dichlorofluorescein diacetate&#160;</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">C369</td>
			<td>Commonly known as cDCFDA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:475px;">9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">L7528</td>
			<td>Commonly known as Lysotracker Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Confocal microscope (e.g. Zeiss LSM 510)</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">ImageJ</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td>Download for free from https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">LB Broth powder</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">22700041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Agar</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">NaCl</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Peptone</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:119px;">S71604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cholesterol powder</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">C3045&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">CaCl2</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">449709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">MgSO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">K3PO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">P5629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Sodium Azide</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">DMSO</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:119px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cover Slips</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">3406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Agarose</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Incubator</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Mirror or other smooth flat surface</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

线虫秀丽线虫(C. 线虫) 是一种广泛用于研究长寿和发育途径的模型系统。这种研究通过动物的透明性、向前和反向基因检测的能力、产生荧光标记蛋白的相对容易性以及荧光染料的使用来促进, 它们可以杏仁到早期胚胎或纳入其食物 (大肠杆菌菌株 OP50) 标签细胞器 (例如9-二乙基-5 H 苯并 (a) phenoxazine-5-一和 (3-{2-[(1 H, 1 ' H-22 '-bipyrrol 5-基 kappaN (1)) methylidene]-2 H-咯-5-基 kappaN}-N-[2-(二甲基氨基) 乙基] propanamidato) (difluoro) 硼)。在这里, 我们提出使用荧光 pH 敏感染料, 染色肠道溶酶体, 提供了动态, 生理变化的溶酶体酸度在活蠕虫的视觉读数。该协议不测量溶酶体 pH 值, 而是旨在建立一个可靠的方法来评估溶酶酸度的生理相关变化。cDCFDA 是一种细胞 permeant 化合物, 转化为荧光荧光 5-(和-6) 羧基-2 ', 7 '-dichlorofluorescein (cDCF) 水解后, 细胞内酯。质子内溶酶体陷阱 cDCF 在这些细胞器中, 在那里积聚。该染料由于其4.8 的低碱性, 已被用作酵母中的 pH 传感器。在这里, 我们描述了使用 cDCFDA 作为食物补充, 以评估肠道溶酶体的酸度在线虫.这种技术允许检测活动物中的碱化溶酶体,并具有广泛的实验应用, 包括对衰老、自噬和溶酶合成的研究。

cDCFDA 渍溶酶在 ph 依赖性的方式, 它的低 pKa 和准备吸收入溶酶使它成为理想的 pH 传感器21。cDCFDA 染色强度与溶酶体 ph 值成反比 (即染色强度随 pH 值的降低而增加)18,22。cDCFDA 信号在动物溶酶体中一直很弱, 在20毫米的氯喹, 一个溶酶体酸化抑制剂, 和在蠕虫耗尽的 v-atp 酶, 蛋白质复合体的膜上的溶酶, 质子?...

Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

用 pH 敏感的生命染料 5 (6)-羧基-2 ', 7 '-dichlorofluorescein 双乙酸 (cDCFDA) 研究了在线虫的肠道中溶酶体酸度损失的分步指南

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

assessing-lysosomal-alkalinization-intestine-live-caenorhabditis

Research

JoVE Journal

Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

5.9K 次观看.  University of Saskatchewan. 用 pH 敏感的生命染料 5 (6)-羧基-2 ', 7 '-dichlorofluorescein 双乙酸 (cDCFDA) 研究了在线虫的肠道中溶酶体酸度损失的分步指南

视频: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

协议中再生的小鼠肠道使用条件潘氏细胞中的作用分析<em&gt;的Cre-LOX</em&gt;小鼠模型

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.