Name: using fluorescent proteins to monitor glycosome dynamics in the african trypanosome
Uploaded: 2014-08-19T13:00:00.000+00:00
Duration: 10 min 4 s
Description: Watch this Scientific Journal Video about Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome at JoVE.com

This work was funded by the Creative Inquiry Program for Undergraduate Research and the Calhoun Honors College at Clemson University.

1，一般锥虫畜牧<ol><li>称重固体为SDM79媒体制剂（ 表1）。 </li><li>保存在4℃的50毫升锥形或保鲜袋包。注:本产品保质期至少为6个月。 </li><li>解冻的胎牛血清（FBS）在37℃水浴，并通过逆周期混合。注:FBS是从供应商的无菌溶液好评。过滤消毒FBS降低其支持寄生虫的生长能力。 </li><li>一旦整个瓶解冻，热灭活血清30分钟，在56℃水浴，定期搅拌，尽量减少血清成分的沉淀。 </li><li>解冻青霉素/链霉素溶液（青霉素/链霉素），血红素（2毫克/毫升在50mM的NaOH），并在室温下基本培养基鹰维生素溶液。 </li><li>到1000量筒，加20.2克SDM79固体（ 表1）的液体成分（ 表2）拌匀上的轰动板。 </li><li>调节pH至7.35，并带来体积至850毫升的水。 </li><li>过滤器通过连接真空软管到滤波器瓶底杀菌媒体。施加真空，直到溶液被过滤。 </li><li>在生物安全柜中取出过滤器上，加入150ml的热灭活胎牛血清。无菌帽和密封介质瓶取下塑料盖。 </li><li>制备SDM80介质中相同的方式SDM79有以下例外;不添加葡萄糖或葡萄糖，并使用MEM无谷氨酰胺。代替150ml的热灭活的FBS，加入135毫升的渗析，热灭活的FBS，和15毫升的热灭活的胎牛血清。 </li><li>培养PCF寄生虫中为25cm 2烧瓶中，用10ml合适的培养基中于27℃和5％CO 2。注:细胞应每天使用血细胞计数或流式细胞仪进行计数（参见第6节），并培养物应保持在密度在1×10 5和5×10 6个细胞/ ml。 </li></ol> 2，Tr的PCF寄生虫ansfection与荧光记者构造注:要遵循glycosome动态，含醛缩酶融合，增强黄色荧光蛋白的过氧化物酶体靶向序列（PTS2）记者蛋白表达的寄生虫。该序列编码所述融合蛋白克隆到pXS2bla 12，其中包含了procyclin子和微管蛋白的基因间区域，其直接同源重组到基因组和杀稻瘟菌素抗性基因用于选择。 Procyclic细胞系携带编码T7聚合酶和四环素阻遏（PF29-13）的基因转化的靶向构建体，pXS2:PTS2eYFP。 <ol><li>通过将表3中所列的组分制备cytomix。筛选在RT消毒和储存。 </li><li>线性化的质粒DNA用MluI通过吸取10微升的DNA（1微克/微升），5微升限制性内切酶缓冲液，33微升的水，和2μ，L的MluI酶到离心管。在37℃下反应1小时。 </li><li>纯化的限制，加入1倍体积的结合缓冲液中，以限制性内切酶消化消化。通过涡旋混合的结合缓冲液和消化的DNA。短暂离心所述管的底部以收集样品。 </li><li>插入DNA结合柱到2 ml收集管中，转移消化的DNA /结合缓冲液列。 </li><li>离心10,000 xg离心1分钟。离心后，弃去收集管中的滤液和列返回到收集管中。 </li><li>加入700μl的SPW洗涤缓冲液和离心机以10,000 xg离心1分钟。 </li><li>从收集管中丢弃滤液，返回列到收集管中，重复SPW洗涤步骤和离心。 </li><li>丢弃滤液，返回柱到收集管中，并离心3分钟，以10,000 XG空柱以除去残余的乙醇。 </li><li>在生物安全柜，扔掉COL经文管处列在无菌的离心管。 </li><li>为了洗脱DNA，加入25微升无菌水至柱并在室温下孵育2分钟。离心机以10,000 xg离心1分钟。 </li><li>添加另一个25μL无菌水在生物安全罩，以列，在室温下孵育2分钟。 </li><li>离心机以10,000 XG的速度，持续1分钟。 </li><li>在生物安全柜的DNA整卷转移到新的无菌离心管中。注:此步骤可以防止污染，从开盖离心过程中。 </li><li>加入无菌的，纯化的线性化的DNA（10微克），以400微升过滤灭菌cytomix的。 </li><li>如第6 5×10 7 -10 8个细胞在室温描述和收获离心800×g离心15分钟计数细胞。 </li><li>倒掉上清液，用1毫升血清吸管重悬细胞沉淀在450微升的DNA + cytomix的。 </li><li>含Ce传输解决方案<html> LLS，DNA和cytomix在电穿孔室的无菌4毫米间隙比色皿和地方杯中。 </li><li>选择"指数衰减"，并手动输入以下设置:电压:1.5千伏，容量:25 MF，电阻:Ω，和比色皿:4毫米。按脉搏。一旦脉冲完成后，从电穿孔室中取出试管，返回到生物安全柜中。 </li><li>到一个新的无菌瓶，吸管10毫升SDM79的。转移转化细胞向烧瓶中，用10ml SDM79的。 </li><li>培育无药物选择24小时，在27℃，5％CO2。 24小时后，加入G418（15微克/毫升），潮霉素（50微克/毫升），杀稻瘟素（10微克/毫升）。通过1毫升转化细胞与药物进入9毫升SDM79的药物。 </li><li>如在第6和7。当细胞是荧光描述的，并已达到1×10 7个/ ml的细胞密度每日分析细胞，使stabilates对于长期贮存（3.1-3.3）。 </li></ol>ve_title"&gt; 3。制作Stabilates <ol><li>使冷冻介质中，添加100％的甘油等体积cytomix和过滤消毒。 </li><li>加入200μl冷冻介质的800微升细胞（1×10 7）在离心管，轻轻混匀，通过反演和放置两个保丽龙架之间，并冻结在-80℃。 </li><li>一旦冷冻（〜24小时），转移细胞到液氮中。注:移动到细胞液氮 24小时后，如再储存于-80是很重要℃，导致细胞活力下降。 </li></ol> 4，冷冻解冻股<ol><li>除去从-80冷冻瓶℃并解冻在室温约10分钟。 </li><li>吸取9毫升SDM79与药物进入一个新的无菌瓶中。添加解冻的细胞以这个烧瓶中。通过将在一个新的烧瓶1ml该培养至9毫升SDM79的（1×10 5个/ ml的终浓度）通过细胞1:10。 </li><li>在细胞达到10 7 / ml的密度，开始设定仪和稀释试验。 </li></ol> 5，流式细胞仪设置<ol><li>打开流式细胞仪，打开CFLOW Plus计划。注意:在运行任何样品之前，流体应根据步骤5.2-5.7被反和漂洗。 </li><li>取代，其中所述SIP存储与空管​​超纯水的管。 </li><li>选择"倒冲"按钮。 </li><li>一旦反吹完成后，从SIP和丢弃去除微量离心管中。 </li><li>将载有关于SIP加入1ml新的超纯水一个新的离心管中。 </li><li>选择新井CFLOW程序。在"运行限制"2分钟的设定时间。在"射流"中选择"快"和"运行"。 </li><li>一旦2分钟的时限完成后，单击"删除样本数据"按钮。 </li></ol>采用流式细胞仪6细胞计数<ol><li>与样品代替水来进行计数。设置"运行限制"到30秒，"射流"到"快"，然后选择"运行"。 </li><li>在30秒的时间限制完成后，CFLOW Plus将提供事件/μL在"上一次运行。"为每一种文化重复计数。注:在细胞达到1×10 7个/ ml，继续稀释法。一个稀释试验完成后电池应停药。细胞更长的时间培养丧失其对环境条件的反应能力。 </li></ol>采用流式细胞仪7测量荧光<ol><li>为了评估荧光，设置"运行限制"10000事件，"射流"到"慢"。选择"设置门槛"。在"小门槛"，选择"永久取消活动的"，"FSC-H"（在下拉框中），然后输入小于30000。点击"应用"，然后"关闭"。 </li><li>选择220;直方图"中的新剧情窗口中的一个按钮，然后选择"FSC-A"从下拉列表中，选择"FL1-A"。注:在530纳米波长FL1-A测量荧光。 </li><li>选择"运行"，然后重复所有文化。 </li><li>运行通过流体线清洁溶液2分钟，水2分钟。 </li></ol> 8稀释测定<ol><li>通过细胞的1×10 5个细胞/ ml在3ml SDM79的密度的25cm 2培养烧瓶中，并通过后立即，然后在3小时和24小时，测量荧光。 </li></ol> 9，数据分析<ol><li>在CFLOW加选择"分析"选项卡。 </li><li>点击下的"创建一个新的阴谋。"选择"FSC-A"和一个下拉列表"直方图"按钮就会出现。选择通道"FL1-A"。 </li><li>突出一个很好分析。选择"门"按钮，手动绘制门感兴趣的人群。 </li><li>流动Plus将提供电池"伯爵"这个门中和"％的该地块"。 </li></ol>

<ol>
	<li>Stuart, 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=Kinetoplastids:+related+protozoan+pathogens,+different+diseases.">Kinetoplastids: related protozoan pathogens, different diseases.</a> J Clin Invest. 118, 1301-1310 (2008).</li><li>Herman, M., Perez-Morga, D., Schtickzelle, N., Michels, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnover+of+glycosomes+during+life-cycle+differentiation+of+Trypanosoma+brucei.">Turnover of glycosomes during life-cycle differentiation of Trypanosoma brucei.</a> Autophagy. 4, 294-308 (2008).</li><li>Herman, M., Gillies, S., Michels, P. A., Rigden, D. J. <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+related+processes+in+trypanosomatids:+insights+from+genomic+and+bioinformatic+analyses.">Autophagy and related processes in trypanosomatids: insights from genomic and bioinformatic analyses.</a> Autophagy. 2, 107-118 (2006).</li><li>Michels, P. A., Bringaud, F., Herman, M., Hannaert, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+functions+of+glycosomes+in+trypanosomatids.">Metabolic functions of glycosomes in trypanosomatids.</a> Biochim Biophys Acta. 1763, 1463-1477 (2006).</li><li>Michels, P. 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=Peroxisomes,+glyoxysomes+and+glycosomes+(review).">Peroxisomes, glyoxysomes and glycosomes (review).</a> Mol Membr Biol. 22, 133-145 (2005).</li><li>Moyersoen, J., Choe, J., Fan, E., Hol, W. G., Michels, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+peroxisomes+and+glycosomes:+trypanosomatid+glycosome+assembly+is+a+promising+new+drug+target.">Biogenesis of peroxisomes and glycosomes: trypanosomatid glycosome assembly is a promising new drug target.</a> FEMS Microbiol Rev. 28, 603-643 (2004).</li><li>Parsons, M., Furuya, T., Pal, S., Kessler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+function+of+peroxisomes+and+glycosomes.">Biogenesis and function of peroxisomes and glycosomes.</a> Molecular and biochemical parasitology. 115, 19-28 (2001).</li><li>Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosomes:+parasites+and+the+divergence+of+peroxisomal+purpose.">Glycosomes: parasites and the divergence of peroxisomal purpose.</a> Mol Microbiol. 53, 717-724 (2004).</li><li>Michels, P. A., Hannaert, V., Bringaud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+aspects+of+glycosomes+in+trypanosomatidae+-+new+data+and+views.">Metabolic aspects of glycosomes in trypanosomatidae - new data and views.</a> Parasitol Today. 16, 482-489 (2000).</li><li>Michels, P. A., Hannaert, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+kinetoplastid+glycosomes.">The evolution of kinetoplastid glycosomes.</a> J Bioenerg Biomembr. 26, 213-219 (1994).</li><li>Hannaert, V., Michels, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+function,+and+biogenesis+of+glycosomes+in+kinetoplastida.">Structure function, and biogenesis of glycosomes in kinetoplastida.</a> J Bioenerg Biomembr. 26, 205-212 (1994).</li><li>Bauer, S., Morris, J. C., Morris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmentally+regulated+glycosome+protein+composition+in+the+african+trypanosome.">Environmentally regulated glycosome protein composition in the african trypanosome.</a> Eukaryot Cell. 12, 1072-1079 (2013).</li><li>Colasante, C., Ellis, M., Ruppert, T., Voncken, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+of+glycosomes+from+bloodstream+form+and+procyclic+culture+form+Trypanosoma+brucei+brucei.">Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei.</a> Proteomics. 6, 3275-3293 (2006).</li><li>Opperdoes, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentation+of+carbohydrate+metabolism+in+trypanosomes.">Compartmentation of carbohydrate metabolism in trypanosomes.</a> Annu Rev Microbiol. 41, 127-151 (1987).</li><li>Kessler, P. S., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+role+of+compartmentation+of+glycolysis+in+procyclic+form+Trypanosoma+brucei:+RNA+interference+studies+of+PEX14,+hexokinase,+and+phosphofructokinase.">Probing the role of compartmentation of glycolysis in procyclic form Trypanosoma brucei: RNA interference studies of PEX14, hexokinase, and phosphofructokinase.</a> The Journal of biological chemistry. 280, 9030-9036 (2005).</li><li>Bringaud, F., Riviere, L., Coustou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+trypanosomatids:+adaptation+to+available+carbon+sources.">Energy metabolism of trypanosomatids: adaptation to available carbon sources.</a> Molecular and biochemical parasitology. 149, 1-9 (2006).</li><li>Coustou, V., 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=Glucose-induced+remodeling+of+intermediary+and+energy+metabolism+in+procyclic+Trypanosoma+brucei.">Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei.</a> The Journal of biological chemistry. 283, 16342-16354 (2008).</li><li>Lamour, N., 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=Proline+metabolism+in+procyclic+Trypanosoma+brucei+is+down-regulated+in+the+presence+of+glucose.">Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose.</a> The Journal of biological chemistry. 280, 11902-11910 (2005).</li><li>Vertommen, D., 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=Differential+expression+of+glycosomal+and+mitochondrial+proteins+in+the+two+major+life-cycle+stages+of+Trypanosoma+brucei.">Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei.</a> Molecular and biochemical parasitology. 158, 189-201 (2008).</li><li>Vickerman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+cycles+and+biology+of+pathogenic+trypanosomes.">Developmental cycles and biology of pathogenic trypanosomes.</a> British medical bulletin. 41, 105-114 (1985).</li><li>Lorenz, P., Maier, A. G., Baumgart, E., Erdmann, R., Clayton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongation+and+clustering+of+glycosomes+in+Trypanosoma+brucei+overexpressing+the+glycosomal+Pex11p.">Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p.</a> The EMBO journal. 17, 3542-3555 (1998).</li><li>Saveria, 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=Conservation+of+PEX19-binding+motifs+required+for+protein+targeting+to+mammalian+peroxisomal+and+trypanosome+glycosomal+membranes.">Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes.</a> Eukaryot Cell. 6, 1439-1449 (2007).</li><li>Banerjee, S. K., Kessler, P. S., Saveria, T., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+trypanosomatid+PEX19:+functional+characterization+reveals+impact+on+cell+growth+and+glycosome+size+and+number.">Identification of trypanosomatid PEX19: functional characterization reveals impact on cell growth and glycosome size and number.</a> Molecular and biochemical parasitology. 142, 47-55 (2005).</li><li>Milagros Camara Mde, L., Bouvier, L. A., Miranda, M. R., Pereira, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+Trypanosoma+cruzi's+glycosomal+adenylate+kinase+containing+a+peroxisomal+targeting+signal.">Identification and validation of Trypanosoma cruzi's glycosomal adenylate kinase containing a peroxisomal targeting signal.</a> Experimental parasitology. 130, 408-411 (2012).</li><li>Wiemer, E. A., Wenzel, T., Deerinck, T. J., Ellisman, M. H., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+peroxisomal+compartment+in+living+mammalian+cells:+dynamic+behavior+and+association+with+microtubules.">Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules.</a> The Journal of cell biology. 136, 71-80 (1997).</li><li>Kim, P. K., Mullen, R. T., Schumann, U., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+maintenance+of+mammalian+peroxisomes+involves+a+de+novo+PEX16-dependent+pathway+from+the+ER.">The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER.</a> The Journal of cell biology. 173, 521-532 (2006).</li><li>Hoepfner, D., Schildknegt, D., Braakman, I., Philippsen, P., Tabak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+endoplasmic+reticulum+to+peroxisome+formation.">Contribution of the endoplasmic reticulum to peroxisome formation.</a> Cell. 122, 85-95 (2005).</li><li>Furuya, 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=Glucose+is+toxic+to+glycosome-deficient+trypanosomes.">Glucose is toxic to glycosome-deficient trypanosomes.</a> Proc Natl Acad Sci U S A. 99, 14177-14182 (2002).</li></ol>

The authors have nothing to disclose.

Glycosomes是必不可少的，动态的，寄生虫特异性的细胞器。调节这些细胞器的生物合成，维持，增殖和重塑的过程可能包括可能被利用于治疗目的的药物靶标。尽管潜在的高丰度这样的药物靶点，glycosome生物合成领域已经落后于其它生物体类似过程的研究落后，主要是由于缺乏一个易于处理的，高吞吐量的系统，通过该监测快速，动态的细胞器中的反应在活细胞中。 荧光的细胞器报告系统已用于研究细胞器动力学在高等真核细胞，如酵母，植物，真菌和哺乳动物23-25。我们已经生成的PCF寄生虫表达PTS2eYFP是针对成熟glycosomes的转基因菌株，产生荧光的细胞器。变化glycosome成分可以通过下面的细胞荧光来监测。使用本系统时，我们已经发现，环境条件，特别是葡萄糖，调节glycosome组合物12。 在对比显微术方法通常用于研究kinetoplastid寄生虫的细胞器动力学，记者系统提供了一个方法，通过它来快速筛选化合物以及影响整体glycosome动力学条件。然而，这些变化中的荧光变化反映在整体细胞器的组合物。进一步的生化和微观实验要求限定的细胞群之间呈现不同的荧光强度的特定分子的差异。 我们已经确定了一些在重塑实验的关键步骤。有趣的是，我们已经发现，当细胞是更长的时间（两个以上道次）中培养，其响应于环境变化的行为是不可预测的。延长培养后，将细胞从高密度通入新鲜米EDIA，表现出朦胧的人口临时增加，这表明它们仍然能够重塑glycosomes，但24小时后死亡。我们还遇到过在没有重塑观察到的情况。这样做的原因行为是不清楚的，但我们已经发现，如这里描述的，当细胞被处理（限制的孔通道的编号，2和维持培养物低于1×10 7个/毫升），glycosome重塑是可重复的。当细胞不再响应于环境条件，retransforming细胞与报道子构建体的质粒通常补救这种情况。 虽然这个系统已被用于研究在非洲锥虫glycosome行为，它也可以通过融合荧光蛋白的氨基酸序列，其直接蛋白质定位到其他细胞区室采用在其它寄生虫的细胞器的研究。

布氏锥虫引起非洲昏睡病在人类和一种消耗性疾病，那加那病，牛。在这些疾病的治疗中使用的药物是陈旧的和非常有毒的，疫苗无法提供，并且对于耐药性的发展潜力就必须寻找新的药物靶标1。 在它的生命周期，T布氏，昆虫载体和哺乳动物宿主之间交替;两台主机，在目前非常不同的环境中的寄生虫必须生存。发生的许多代谢和形态的变化，因为寄生虫暴露于不同的环境条件。其中最显着的变化是单膜界的寄生虫特异性微体观察，称为glycosomes 13。 血糖水平相对较高（约5毫米）的血液和血液寄生虫（BSF）通过糖酵解WH产生ATP的专ILE线粒体代谢被抑制14。不像其它真核生物中，糖酵解发生在细胞质中，T.布氏最间隔化的glycosomes 14,15糖酵解酶。 à吸血过程中的寄生虫就由采采蝇和体验葡萄糖下降，其中下降到检测不到的水平之内被摄入的飞行15分钟。昆虫的新陈代谢，procyclic形式（PCF），寄生虫是更灵活和葡萄糖，以及氨基酸，如脯氨酸，可以在ATP 16-18的合成中使用。比较蛋白质组学研究揭示生命周期中glycosomal和依赖性变化线粒体蛋白与糖蛋白在血液中的寄生虫增加，参与TCA循环和呼吸链13,19线粒体蛋白。虽然许多研究都集中在BSF和PCF glycosomes之间的差异，知之甚少发生响应于被膜中的PCF glycosomes的变化ironmental变化。 在苍蝇的肠，血糖水平低，瞬间增加在喂食20。在大多数在体外研究中，PCF寄生虫生长在含葡萄糖的介质。然而，最近的研究已经表明，在反应显著PCF代谢变化成葡萄糖的可用性17。在没有葡萄糖，脯氨酸摄取和脯氨酸脱氢酶活性增加了18。这种变化在线粒体代谢很可能伴随着glycosome组成和形态的变化，但是，这并没有被直接评估。 电子和荧光显微镜是用来共同技术研究glycosome动力学T。布氏 2,21-24。这些协议是时间和劳动力密集型的，昂贵的，并且难以适应实时研究和高通量的协议。为了克服这个限制，荧光细胞器报告系统Used来研究哺乳动物和酵母细胞器系统已被修改用于T。布氏 12。 荧光的细胞器报告系统已被广泛用于在高等真核生物如酵母，植物和哺乳动物细胞25-27。在这样的系统中，荧光蛋白融合到靶向蛋白质到特定细胞器的氨基酸序列。靶蛋白的降解或合成是通过荧光测定和改变的细胞器组合物是通过改变细胞的荧光反射。 当增强型黄色荧光蛋白（EYFP）的开放读框融合至II型过氧化物酶体靶向序列（PTS2） 如图12所示 ，PTS2eYFP蛋白质导入到成熟，导入感受态glycosomes和荧光可以通过流式细胞仪进行监测。在glycosome组成变化是通过改变细胞的荧光反射。这个系统可以在甲阶辅助咏调控环境引起的变化glycosome组成的机制。 此手稿描述了一种glycosome报告系统在PCF中的寄生虫的产生结合流式细胞术监测活寄生虫实时glycosome动力学，并提供如何被用于跟踪响应于不同的环境变化glycosome组合物的例子。总之，glycosome组合物是由胞外葡萄糖浓度和对数相培养的通路影响到新鲜培养基触发变化glycosome组合物。此系统可以被修改以研究在锥虫及其它寄生虫其它细胞器的动态行为。

<table><tbody><tr><td>Adenosine</td><td>Avocado Research Chemicals Ltd</td><td>A10781</td><td>SDM79 Ingredient</td></tr><tr><td>L-Alanine</td><td>Avocado Research Chemicals Ltd</td><td>A15804</td><td>SDM79 Ingredient</td></tr><tr><td>L-Arginine</td><td>CalBiochem</td><td>1820</td><td>SDM79 Ingredient</td></tr><tr><td>p-Aminobenzoic acid</td><td>ICN Biomedicals</td><td>102569</td><td>SDM79 Ingredient</td></tr><tr><td>Basal Medium Eagle Vitamin Solution (100x)</td><td>Sigma</td><td>B6891</td><td>SDM79 Ingredient</td></tr><tr><td>Biotin</td><td>Fisher</td><td>BP232-1</td><td>SDM79 Ingredient</td></tr><tr><td>Calcium chloride</td><td>VWR</td><td>BDH0224</td><td>Cytomix</td></tr><tr><td>EDTA</td><td>Fisher</td><td>S311-100</td><td>Cytomix ingredient</td></tr><tr><td>EZNA Gel Extraction kit</td><td>Omega Biotek</td><td>D2500-01</td><td>DNA purifiation</td></tr><tr><td>Research grade Serum</td><td>Fisher</td><td>03-600-511</td><td>SDM79 Ingredient</td></tr><tr><td>Folic acid</td><td>ICN Biomedicals</td><td>101725</td><td>SDM79 Ingredient</td></tr><tr><td>Glucosamine HCl</td><td>ICN Biomedicals</td><td>194671</td><td>SDM79 Ingredient</td></tr><tr><td>Glucose</td><td>GIBCO</td><td>15023-021</td><td>SDM79 Ingredient</td></tr><tr><td>L-Glutamine</td><td>CalBiochem</td><td>3520</td><td>SDM79 Ingredient</td></tr><tr><td>Glycerol</td><td>Acros Organics</td><td>Ac15892-0010</td><td>Freezing media</td></tr><tr><td>Graces insect cell media powder</td><td>GIBCO</td><td>11300-043</td><td>SDM79 Ingredient</td></tr><tr><td>Hemin</td><td>MP Biomedicals</td><td>194025</td><td>SDM79 Ingredient</td></tr><tr><td>Guanosine</td><td>Avocado Research Chemicals Ltd</td><td>A11328</td><td>SDM79 Ingredient</td></tr><tr><td>HEPES</td><td>MP Biomedicals</td><td>194025</td><td>SDM79 Ingredient</td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td>Cytomix ingredient</td></tr><tr><td>L-Methionine</td><td>Fisher</td><td>BP388-100</td><td>SDM79 Ingredient</td></tr><tr><td>MEM Amino Acids (50x)</td><td>Cellgro</td><td>25-030-CI</td><td>SDM79 Ingredient</td></tr><tr><td>NEAA Mixture (100x)</td><td>Lonza</td><td>13-114E</td><td>SDM79 Ingredient</td></tr><tr><td>Minimal Essential Medium (1x) with L-glutamine</td><td>Cellgro</td><td>10-010-CM</td><td>SDM79 Ingredient</td></tr><tr><td>MOPS</td><td>Fisher</td><td>BP308-500</td><td>SDM79 Ingredient</td></tr><tr><td>Sodium biocarbonate</td><td>Fisher</td><td>S233-500</td><td>SDM79 Ingredient</td></tr><tr><td>Penicillin-streptomycin Solution</td><td>Cellgro</td><td>30-002-CI</td><td>SDM79 Ingredient</td></tr><tr><td>L-Phenylalanine</td><td>ICN Biomedicals</td><td>102623</td><td>SDM79 Ingredient</td></tr><tr><td>Potassium chloride</td><td>Fisher</td><td>P217-500</td><td>Cytomix ingredient</td></tr><tr><td>Potassium phosphate dibasic anhydrous</td><td>Fisheer</td><td>P290-212</td><td>Cytomix ingredient</td></tr><tr><td>L-Proline</td><td>Fisher</td><td>BP392-100</td><td>SDM79 Ingredient</td></tr><tr><td>L-Serine</td><td>Acros Organics</td><td>56-45-1</td><td>SDM79 Ingredient</td></tr><tr><td>Pyruvic acid, sodium salt</td><td>Acros Organics</td><td>113-24-6</td><td>SDM79 Ingredient</td></tr><tr><td>L-Taurine</td><td>TCI America</td><td>A0295</td><td>SDM79 Ingredient</td></tr><tr><td>L-Threonine</td><td>Acros Organics</td><td>72-19-5</td><td>SDM79 Ingredient</td></tr><tr><td>L-Tyrosine</td><td>ICN Biomedicals</td><td>103183</td><td>SDM79 Ingredient</td></tr><tr><td>E.Z.N.A. Cycle Pure kit</td><td>Omega Biotek</td><td>D6492-02</td><td>DNA purification</td></tr><tr><td>Binding buffer</td><td>Omega Biotek</td><td>PDR041</td><td>DNA purification</td></tr><tr><td>SPW wash buffer</td><td>Omega Biotek</td><td>PDR045</td><td>DNA purification</td></tr><tr><td>Gene Pulser Xcell</td><td>Biorad</td><td>165-2660</td><td>Trypanosome transformation</td></tr><tr><td>4 mm Electroporation cuvettes</td><td>VWR</td><td></td><td>Trypanosome transformation</td></tr></tbody></table>

using fluorescent proteins to monitor glycosome dynamics in the african trypanosome

布氏锥虫是一种kinetoplastid寄生虫引起非洲人类锥虫病（HAT），或昏睡病和消耗性疾病，那加那病，牛1。哺乳动物宿主的血流和采采蝇矢量之间的寄生虫交替。许多细胞器的组合物，以响应这些不同的胞外条件2-5改变。 Glycosomes是高度专业化的过氧化物酶体中，许多参与糖酵解的酶是条块。在发展和环保监管的方式4-11 Glycosome成分的变化。目前，用于研究glycosome动力学中最常见的技术是电子和荧光显微镜;技术是昂贵，费时费力，而且不容易适用于高通量分析。 为了克服这些限制，荧光-glycosome报道系统中该增强型黄色荧光蛋白（EYFP）融合于过氧化物酶体靶向序列（PTS2），其引导融合蛋白glycosomes 12，已经建立。经PTS2eYFP融合蛋白的进口，glycosomes成为荧光。细胞器的降解和回收导致的​​荧光，可以通过流式细胞术测量的损失。大量的细胞（5000个细胞/秒）可在实时无需大量的样品制备待分析诸如固定和装配。这种方法提供了检测响应波动的环境条件变化的细胞器组成的快速路。

在这个系统中，观察到在glycosome组合物中的葡萄糖依赖性变化。当细胞生长在含葡萄糖的介质，两个总体观察;一个亮点，一个朦胧（ 图2A）。昏暗的细胞怀有不成熟glycosomes，并未进口PTS2eYFP而明亮的细胞怀有成熟和未成熟glycosomes 12的混合物。当葡萄糖是存在于所述介质，glycosome蛋白的错误定位是致死15,28和glycosome蛋白表达可能紧密进口耦合。这种紧密调控负责暗淡的细胞，其中，当所述细胞缺乏足够的导入功能glycosome蛋白表达被抑制的外观。一旦glycosome进口机器完全组装和功能，细胞表达glycosome蛋白质，然后将其导入到glycosomes，产生荧光的细胞。在葡萄糖耗尽的媒体，glycosome蛋白质的错误定位是不能容忍ð15和双峰人口分布被替换为具有更广泛的荧光（ 图2B）的单峰。 此系统已被用于标识触发变化glycosome组合物的条件。当含有〜10％的昏暗的细胞（ 图3A），高密度培养物中通入新鲜培养基中，有在昏暗的细胞（ 图3B）的百分比暂时升高。通过24小时。原始人群分布重新建立（ 图3C）。 <table border="0" cellpadding="0" cellspacing="0" ><tbody><tr ><td > SDM79固体 </td><td > 重量（g / L） </td></tr><tr ><td >美惠昆虫细胞培养基粉末</td><td> 2 </td></tr><tr ><td >血糖</td><td> 1 </td></tr><tr ><td > HEPES </td><td> 8 </td></tr><tr ><td >公开资讯观测站</td><td> 5 </td></tr><tr ><td > 碳酸氢钠 </sub&gt; </td><td> 2 </td></tr><tr ><td >丙酮酸钠</td><td> 0.1 </td></tr><tr ><td > L-丙氨酸</td><td> 0.2 </td></tr><tr ><td > L-精氨酸</td><td> 0.1 </td></tr><tr ><td > L-谷氨酰胺</td><td> 0.3 </td></tr><tr ><td > L-DL - 蛋氨酸</td><td> 0.07 </td></tr><tr ><td > L-苯丙氨酸</td><td> 0.08 </td></tr><tr ><td > L-脯氨酸</td><td> 0.6 </td></tr><tr ><td > L-丝氨酸</td><td> 0.06 </td></tr><tr ><td > L-牛磺酸</td><td> 0.16 </td></tr><tr ><td > L-苏氨酸</td><td> 0.35 </td></tr><tr ><td > L-酪氨酸</td><td> 0.1 </td></tr><tr ><td >腺苷</td><td> 0.01 </td></tr><tr ><td >鸟苷</td><td> 0.01 </td></tr><tr ><td >氨基葡萄糖盐酸盐</td><td> 0.05 </td></tr><tr ><td >叶酸</td><td> 0.004 </td></tr><tr ><td > R-氨基苯甲酸</td><td> 0.002 </td></tr><tr ><td >生物素</td&gt; <td> 0.0002 </td></tr></tbody></table> 表1 SDM79固体组分上。固体培养基组分和量（克/升）被提供。 <table border="0" cellpadding="0" cellspacing="0" ><tbody><tr ><td > SDM79 </td><td ></td></tr><tr ><td >灰熊谷氨酰胺</td><td > 600毫升</td></tr><tr ><td >笔/链球菌</td><td > 10毫升</td></tr><tr ><td > BME维生素解决方案</td><td > 10毫升</td></tr><tr ><td >灰熊氨基酸液</td><td > 8毫升</td></tr><tr ><td > MEM非必需氨基酸溶液</td><td > 6毫升</td></tr><tr ><td >民</td><td> 3.75 </td></tr><tr ><td >水</td><td > 162.25毫升</td></tr></tbody></table> 表2 SDM79液体成分。卷毫升给出。 <table border="0" cellpadding="0" cellspacing="0" ><tbody><tr ><td ><strong&gt;的Cytomix </td><td >对于20毫升</td></tr><tr ><td > 120毫米氯化钾</td><td > 1.4毫升（1μM） </td></tr><tr ><td > 0.15毫米氯化钙2 </td><td > 3毫升（1μM） </td></tr><tr ><td >的10mM K 2 HPO 4 </td><td > 400毫升（0.5M） </td></tr><tr ><td > 25毫米肝素钠</td><td > 500毫升（1μM） </td></tr><tr ><td > 2毫米EDTA </td><td > 80毫升（0.5M） </td></tr><tr ><td > 5毫米氯化镁2 </td><td >百毫升（1μM） </td></tr><tr ><td >水</td><td> 16.52 </td></tr></tbody></table> 表3 Cytomix配方。 <img alt="图1" fo:content-width="5in" width="500" src="/files/ftp_upload/51647/51647fig1highres.jpg" /> 图1荧光glycosome报告系统。 A）PTS2eYFP表达构建一体化。 PCF锥虫转化与对PXS2PTS2eYFP质粒线性化，用限制性酶MluI位。这种结构通过同源重组进入微管蛋白基因（浴池）和PARP序列（PARP），集成了包括驱动PTS2eYFP和组成性表达所需的RNA加工，B中的顺序）PTS2eYFP进口的启动子。 PTS2eYFP合成在其结合的可溶性受体，PEX7，它提供的报告蛋白的glycosomes细胞质中。一旦递送至glycosome膜，所述蛋白质是进口的。成熟的细胞器含进口机械进口PTS2eYFP和荧光而那些不含有功能性进口机器仍然暗淡。 <img alt="图2" fo:content-width="5in" width="500" src="/files/ftp_upload/51647/51647fig2highres.jpg" /> 图2:葡萄糖依赖性glycosome组合物。PCF细胞生长于SDM79（+ GLC）或SDM80（-Glc）和流式细胞仪分析。 10000事件直方图。在SDM79细胞生长的）分析一致表明两个峰。荧光细胞怀有成熟和未成熟的细胞器与具有较为成熟的glycosomes更高的荧光强度的细胞混合物。在SDM79，glycosome蛋白质的错误定位是致命和glycosome蛋白表达和进口必须严格控制。这反映在无细胞的中间荧光强度。 乙）在SDM80的，glycosome蛋白的错误定位是可以容忍的，双峰的人口分布丢失了，并且中间荧光的细胞被观察到。 <img alt="图3" fo:content-width="5in" width="500" src="/files/ftp_upload/51647/51647fig3highres.jpg" /> 图3 Glycosome重塑。glycosomes的重塑是由通道进入新鲜培养基触发。 <st荣&gt; A）直方图在开始培养10000个事件（〜4×10 6个 / ml），B）3小时稀释到新鲜SDM79后有增加在昏暗的细胞不成熟glycosomes落左闸ç内的比例24小时后）直方图稀释文化。 24小时后，人口分布已恢复正常，因为不成熟glycosomes现在包含必要进口的荧光蛋白过氧化物酶体蛋白。

Watch this Scientific Journal Video about Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome at JoVE.com

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

using-fluorescent-proteins-to-monitor-glycosome-dynamics-african

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.9K 次观看.  Clemson University Eukaryotic Pathogens Innovation Center. Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

视频: Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）用于监视淋巴细胞增殖

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

科研

免疫与感染

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

使用<em&gt;在体内</em&gt;影像监测进展的实验小鼠巨细胞病毒感染的新生儿

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

生物学

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

基于 pHluorin2 的布氏锥虫糖体 pH 传感器的开发

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

作者聚焦：使用 pHluorin2 生物传感器监测 布氏锥 虫糖体 pH 值的进展

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

测量布氏活锥虫的动态糖体 pH 值变化

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

利用荧光蛋白监视非洲锥虫Glycosome动态

摘要

探索更多视频

此视频中的章节

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）用于监视淋巴细胞增殖

的视觉检测，以监测T6SS介导的细菌竞争

使用

生物发光成像检测到非洲锥虫病的晚期感染

高通量基因定位在

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

内质网子域的培养细胞中的可视化

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化