The document summarizes a study comparing the effects of Taxol, 10-deacetylbaccatinIII (10-DAB), and BaccatinIII (BacIII) on caveolae dynamics in HeLa cells expressing GFP-Caveolin-1. The key findings are:
1) Taxol treatment caused a transient recruitment of Caveolin-1 to the cell surface followed by internalization, while 10-DAB increased the "kiss and run" dynamics of caveolae.
2) Sustained Caveolin-1 phosphorylation was observed with both Taxol and 10-DAB treatments, with Taxol also inducing phosphorylation of Raf-1, ERK1/2,
Temp-Sensitive Inhibition of Development in Dictyostelium - Dev Bio 251 18-26...James Silverman
1) The Dictyostelium mutant HSB1 is temperature-sensitive for development, aggregating and forming fruiting bodies below 18°C but not above.
2) HSB1 cells have a defective G protein-linked adenylyl cyclase that is not stimulated by GTPγS in vitro but can be rescued by adding wild-type cytosol.
3) Transfection with the wild-type piaA gene rescued the HSB1 mutant phenotype, and sequencing revealed a point mutation in the HSB1 piaA gene resulting in a single amino acid change.
The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
This document summarizes an experiment optimizing expression of G-protein coupled receptors (GPCRs) in HEK293 cells using a bioreactor. The researchers found that using a bioreactor allowed for higher cell densities and expression levels of over 1 mg of recombinant GPCR per liter of culture, compared to 0.1-0.2 mg/L using spinner flasks. A large-scale run in the bioreactor produced 10 mg of purified, bioactive recombinant GPCR. The bioreactor setup improved expression levels for structural characterization of this important class of membrane proteins.
Set7/9 is a lysine methyltransferase that interacts with the transcription factor Pdx1. This study found that:
1) Set7/9 methylates two lysine residues (Lys-123 and Lys-131) on Pdx1. Methylation only occurred with full-length Pdx1, indicating structural requirements.
2) Lys-131 methylation by Set7/9 augments Pdx1's transcriptional activity in luciferase reporter assays.
3) Mice with beta-cell specific deletion of Set7/9 (SetΔβ mice) showed glucose intolerance and impaired insulin secretion from islets, similar to Pdx1-deficient mice. Target genes
This document summarizes the expression of recombinant β-lactoglobulin (rBLG) in prokaryotic and eukaryotic cells. In Escherichia coli, rBLG was expressed with a pET26 vector and was found predominantly in a denatured form, even when expressed in the periplasm. In eukaryotic cells like COS-7 and mouse tibialis muscle, rBLG was expressed in its native conformation. The authors quantified rBLG expression using immunoassays that distinguish between native and denatured rBLG. They found higher expression levels and native folding of rBLG in eukaryotic systems compared to prokaryotic expression
Kupffer Cells Mediate Leptin-Induced Liver Fibrosis.
GASTROENTEROLOGY 2009;137:713–723
JIANHUA WANG,* ISABELLE LECLERCQ,‡ JOANNE M. BRYMORA,* NING XU,* MEHDI RAMEZANI–MOGHADAM,* ROSLYN M. LONDON,* DAVID BRIGSTOCK,§ and JACOB GEORGE*
*Storr Liver Unit, Westmead Millennium Institute, University of Sydney and Westmead Hospital, Westmead, Australia; ‡Laboratory of Gastroenterology, Faculty of
Medicine, Université Catholique de Louvain, Brussels, Belgium; and §Center for Cell and Vascular Biology, Children’s Research Institute, Columbus, Ohio
瘦素(Leptin)是一由脂肪細胞(Adipocyte)所分泌之荷爾蒙,是調控體重及新陳代謝之重要因子。過去研究發現病態肥胖(Obese)、脂肪肝(Nonalcoholic steatohepatitis)及酒精性肝炎(Alcoholic liver disease)等病患之血液循環中,Leptin量有明顯增加。而近期研究報告指出leptin具有促進肝臟纖維化(Liver fibrosis)之能力,當中分子機理並未明確。
在肝纖維化過程中,肝臟星狀細胞(HSC)會被活化增生及促進胞外基質(ECM)產生,而鄰近之Kupffer細胞(KC)則已知可透過促發炎因子(Proinflammatory factor)和促纖維化因子(Profibrogenic factors)例如TGF-β1和ROS影響HSC表現。雖然HSC是肝纖維化過程中重要角色,前人研究卻發現leptin似對HSC無任何調控作用。故本篇作者針對Leptin是否透過間接作用於HSC鄰近之KC,刺激其產生促纖維化因子,以活化HSC。
為探討leptin直接或間接影響HSC之分子機理,本篇作者透過RT-PCR、Immunoblot等分子生物學方法,分別測定leptin刺激後HSC及KC中Collagen I、TIMP1等促纖維化因子基因及蛋白表現,發現leptin雖可促使HSC增生,但對其纖維化能力之影響甚微。而leptin可刺激KC中TGF-β1及CTGF/CCN2等肝纖維化中重要之cytokines表現。另發現Leptin-treated KC-conditioned培養液可刺激HSC增生及增加其中Collagen I、TIMP1等表現,得出了leptin是透過刺激KC來活化HSC之推論。作者亦於後續實驗中,透過磷酸化測定、EMSA等方法探討leptin訊號傳遞作用,發現leptin可活化KC中STAT3、ERK1/2、AKT等路徑,及下游因子AP-1、NF-κB,而此兩種蛋白具有增強TGF-β1及CTGF/CCN2基因表現之能力。
B-box proteins in plants bbx family of plant transcription factorsOm Prakash Patidar
This document summarizes research on B-box proteins. It begins by describing the structure of zinc finger domains and their ability to bind DNA, RNA, or proteins. It then discusses B-box (BBX) proteins, which contain zinc finger domains and are involved in plant protein-protein interactions and transcription factor activity. The document reviews studies of BBX proteins in animals and plants, describing their roles in processes like photomorphogenesis, flowering time regulation, and shade avoidance responses. Case studies are presented on specific BBX proteins in Arabidopsis and crops like rice and soybean.
Temp-Sensitive Inhibition of Development in Dictyostelium - Dev Bio 251 18-26...James Silverman
1) The Dictyostelium mutant HSB1 is temperature-sensitive for development, aggregating and forming fruiting bodies below 18°C but not above.
2) HSB1 cells have a defective G protein-linked adenylyl cyclase that is not stimulated by GTPγS in vitro but can be rescued by adding wild-type cytosol.
3) Transfection with the wild-type piaA gene rescued the HSB1 mutant phenotype, and sequencing revealed a point mutation in the HSB1 piaA gene resulting in a single amino acid change.
The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
This document summarizes an experiment optimizing expression of G-protein coupled receptors (GPCRs) in HEK293 cells using a bioreactor. The researchers found that using a bioreactor allowed for higher cell densities and expression levels of over 1 mg of recombinant GPCR per liter of culture, compared to 0.1-0.2 mg/L using spinner flasks. A large-scale run in the bioreactor produced 10 mg of purified, bioactive recombinant GPCR. The bioreactor setup improved expression levels for structural characterization of this important class of membrane proteins.
Set7/9 is a lysine methyltransferase that interacts with the transcription factor Pdx1. This study found that:
1) Set7/9 methylates two lysine residues (Lys-123 and Lys-131) on Pdx1. Methylation only occurred with full-length Pdx1, indicating structural requirements.
2) Lys-131 methylation by Set7/9 augments Pdx1's transcriptional activity in luciferase reporter assays.
3) Mice with beta-cell specific deletion of Set7/9 (SetΔβ mice) showed glucose intolerance and impaired insulin secretion from islets, similar to Pdx1-deficient mice. Target genes
This document summarizes the expression of recombinant β-lactoglobulin (rBLG) in prokaryotic and eukaryotic cells. In Escherichia coli, rBLG was expressed with a pET26 vector and was found predominantly in a denatured form, even when expressed in the periplasm. In eukaryotic cells like COS-7 and mouse tibialis muscle, rBLG was expressed in its native conformation. The authors quantified rBLG expression using immunoassays that distinguish between native and denatured rBLG. They found higher expression levels and native folding of rBLG in eukaryotic systems compared to prokaryotic expression
Kupffer Cells Mediate Leptin-Induced Liver Fibrosis.
GASTROENTEROLOGY 2009;137:713–723
JIANHUA WANG,* ISABELLE LECLERCQ,‡ JOANNE M. BRYMORA,* NING XU,* MEHDI RAMEZANI–MOGHADAM,* ROSLYN M. LONDON,* DAVID BRIGSTOCK,§ and JACOB GEORGE*
*Storr Liver Unit, Westmead Millennium Institute, University of Sydney and Westmead Hospital, Westmead, Australia; ‡Laboratory of Gastroenterology, Faculty of
Medicine, Université Catholique de Louvain, Brussels, Belgium; and §Center for Cell and Vascular Biology, Children’s Research Institute, Columbus, Ohio
瘦素(Leptin)是一由脂肪細胞(Adipocyte)所分泌之荷爾蒙,是調控體重及新陳代謝之重要因子。過去研究發現病態肥胖(Obese)、脂肪肝(Nonalcoholic steatohepatitis)及酒精性肝炎(Alcoholic liver disease)等病患之血液循環中,Leptin量有明顯增加。而近期研究報告指出leptin具有促進肝臟纖維化(Liver fibrosis)之能力,當中分子機理並未明確。
在肝纖維化過程中,肝臟星狀細胞(HSC)會被活化增生及促進胞外基質(ECM)產生,而鄰近之Kupffer細胞(KC)則已知可透過促發炎因子(Proinflammatory factor)和促纖維化因子(Profibrogenic factors)例如TGF-β1和ROS影響HSC表現。雖然HSC是肝纖維化過程中重要角色,前人研究卻發現leptin似對HSC無任何調控作用。故本篇作者針對Leptin是否透過間接作用於HSC鄰近之KC,刺激其產生促纖維化因子,以活化HSC。
為探討leptin直接或間接影響HSC之分子機理,本篇作者透過RT-PCR、Immunoblot等分子生物學方法,分別測定leptin刺激後HSC及KC中Collagen I、TIMP1等促纖維化因子基因及蛋白表現,發現leptin雖可促使HSC增生,但對其纖維化能力之影響甚微。而leptin可刺激KC中TGF-β1及CTGF/CCN2等肝纖維化中重要之cytokines表現。另發現Leptin-treated KC-conditioned培養液可刺激HSC增生及增加其中Collagen I、TIMP1等表現,得出了leptin是透過刺激KC來活化HSC之推論。作者亦於後續實驗中,透過磷酸化測定、EMSA等方法探討leptin訊號傳遞作用,發現leptin可活化KC中STAT3、ERK1/2、AKT等路徑,及下游因子AP-1、NF-κB,而此兩種蛋白具有增強TGF-β1及CTGF/CCN2基因表現之能力。
B-box proteins in plants bbx family of plant transcription factorsOm Prakash Patidar
This document summarizes research on B-box proteins. It begins by describing the structure of zinc finger domains and their ability to bind DNA, RNA, or proteins. It then discusses B-box (BBX) proteins, which contain zinc finger domains and are involved in plant protein-protein interactions and transcription factor activity. The document reviews studies of BBX proteins in animals and plants, describing their roles in processes like photomorphogenesis, flowering time regulation, and shade avoidance responses. Case studies are presented on specific BBX proteins in Arabidopsis and crops like rice and soybean.
This document summarizes a study on the heterologous expression and characterization of the c1 dioxygenase enzyme from Tetranychus urticae. Key points:
- The c1 dioxygenase gene was inserted into a plasmid and transformed into E. coli cells for expression. However, initial expression attempts did not show overexpression of the protein.
- Additional expression trials were conducted by varying culture conditions and bacterial clones. SDS-PAGE analysis showed some potential overexpression in new clones induced overnight at 37°C, though further optimization is still needed.
- Once expression is optimized, the goal is to purify the recombinant protein using its His-tag and proceed with structural characterization through crystallization,
This study aims to generate fusion-defective mutants of Chlamydomonas reinhardtii through random insertional mutagenesis using glass bead transformation. Transformed cells expressing paromomycin resistance are selected and screened for inability to mate. Several mating-deficient clones have been identified that are defective in swimming, agglutination or both. Further analysis will determine if any clones possess a fusion defect and identify the insertion location to study genes involved in fusion.
This paper describes a regulatory pathway controlling expression of Borrelia burgdorferi OspC and DbpA proteins. The study found that in B. burgdorferi strain 297, the alternative sigma factor RpoN controls expression of the alternative sigma factor RpoS. RpoS then governs expression of the outer surface lipoproteins OspC and DbpA. This regulatory network was determined through targeted gene disruption of rpoN and rpoS, followed by genetic complementation. The findings provide insight into key regulatory networks that impact B. burgdorferi pathogenesis, host range, and virulence.
This document summarizes a study investigating the oncoprotein EVI1 and its regulation of the carbonic anhydrase 3 (CA3) gene. Microarray analysis identified CA3 as a major target of EVI1. The study aims to validate changes in CA3 expression and determine the mechanism and significance of EVI1-mediated transcriptional regulation of CA3. Results show that EVI1 suppresses CA3 expression at both the mRNA and protein levels by decreasing CA3 promoter activity. This suppression of the antioxidant CA3 gene leads to increased caspase activity and apoptosis in cancer cells expressing EVI1.
1) The protein SAMP1 localizes to both the endoplasmic reticulum (ER) and microtubules, suggesting it may associate the ER with microtubules.
2) RNAi treatments to stop SAMP1 synthesis in HeLa cells resulted in changes to cell morphology, including the presence of stress fibers and abnormal mitotic cell shapes.
3) This indicates SAMP1 likely plays a role in cell adhesion and cytoplasmic structure.
- The caveolin scaffolding domain was first identified and termed in 1996. It is a 20 amino acid region within caveolin that interacts with signaling proteins like Src kinases.
- The caveolin scaffolding domain negatively regulates the activity of Src kinases and other signaling molecules by preferentially interacting with their inactive conformations.
- This scaffolding domain is important for caveolin oligomerization and organization of "pre-assembled signaling complexes" within caveolae membrane domains.
This study aims to analyze the distribution of 5-hydroxymethylcytosine (5-hmC) in the hippocampus of an Alzheimer's mouse model compared to healthy mice. DNA will be isolated from the hippocampus and analyzed using a microarray containing over 20,000 promoters and 15,000 CpG islands. Antibodies specific to 5-methylcytosine and 5-hmC will isolate DNA fragments containing these modifications, which will then be amplified and compared between the transgenic and healthy mice to assess epigenetic changes associated with Alzheimer's Disease. The results are expected to show increases, decreases, or no change in 5-hmC levels in the transgenic mouse model compared to controls.
- The study investigated the effect of calcium chloride concentration on the transformation efficiency of E. coli with plasmids pUC19 and pBR322, which differ in size.
- Maximum transformation efficiency was observed at 0.15M CaCl2 for pUC19 and 0.1M CaCl2 for the larger pBR322 plasmid.
- Increasing calcium chloride concentration above these levels decreased transformation efficiency for both plasmids, with no transformants observed above 0.2M, possibly due to decreased cell viability in hypertonic conditions.
ReedWoyda_Introducing Green Fluorescence Into Homo sapiens And Escherichia Co...Reed Woyda
This study aimed to introduce the green fluorescent protein (GFP) gene into E. coli and human cells. GFP was successfully inserted into E. coli and shown to be expressed under control of the L-arabinose promoter. Addition of restriction sites to GFP was also successful, allowing for digestion of the GFP and pcDNA plasmid. However, ligation of the digested GFP fragment into pcDNA was unsuccessful, likely due to nuclease contamination. Expression of GFP in human cells could not be verified due to a technical error during immunoblotting. While some goals were achieved, such as GFP expression in E. coli, further optimization is needed to fully introduce GFP into human cells via this methodology.
A colony to-lawn method for efficient transformation of escherichia coliCAS0609
This document describes a new method for efficiently transforming Escherichia coli called the "colony-to-lawn" method. The key steps are: 1) lysing plasmid-containing donor cells to release plasmids, 2) scraping recipient cells directly from a lawn on an agar plate into the lysate, allowing transformation without preparing competent cells. This method saves time compared to traditional methods by skipping plasmid purification and competent cell preparation. It was also used to conveniently screen positive clones after DNA ligation and transformation during construction of a mutant library.
This document discusses phospholipase C (PLC) and its role in neutrophil degranulation and T cell cytotoxicity. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. PLC signaling is required for lytic granule polarization and effective T cell killing through T cell antigen receptor activation. Neutrophil degranulation involves increases in calcium and PLC production of phosphatidylinositol, which is essential for granule exocytosis. Different intracellular signaling pathways regulate the release of primary, secondary, and tertiary granules from neutroph
This document summarizes research investigating the biosynthesis and processing of succinate dehydrogenase (SDH) subunits in cultured mammalian cells. The key points are:
1. Antisera were produced against purified bovine heart SDH and its large and small subunits, which detected precursor and mature forms of the subunits in rat, pig, and bovine cell lines.
2. In pig kidney cells, newly synthesized precursors of the large and small SDH subunits were detected that were 1000-2000 and 4000-5000 Da larger than the mature forms, respectively.
3. Pulse-chase experiments showed the precursor forms were fully processed to the mature subunits within 45 minutes when uncouplers of
The document discusses research on using phospholipase A2 (PLA2) antibodies to protect cells from radiation damage. PLA2 is an enzyme that releases fatty acids from phospholipids in cell membranes. Exposure to ionizing radiation activates PLA2, leading to cell death. The research proposal is to test whether antibodies that block PLA2 can inhibit this activation and thereby reduce the toxic effects of radiation exposure and increase radiation survival. Blocking PLA2 with antibodies may provide a new radiation protection strategy and tool for diagnosing and monitoring acute radiation sickness.
The document discusses several key topics related to DNA and its role as the genetic material:
- Avery, MacLeod and McCarty's 1944 experiments demonstrated that the transforming principle in bacteria was DNA, establishing it as the genetic material.
- Hershey and Chase's 1967 experiments provided further evidence by showing that DNA, not protein, enters the host cell during bacterial infection.
- Gurdon's 1968 work showed that DNA from specialized cells can reprogram other cells, demonstrating DNA's role in determining cell specialization.
The document contains figures illustrating various aspects of DNA structure, replication, repair, and its processing and packaging in the cell nucleus.
1) The study found that IKKα, like IKKβ and NEMO/IKKγ, is required for the activation of NF-κB target genes in response to TNFα and IL-1 stimulation in mouse embryonic fibroblasts.
2) DNA microarray analysis identified many known and novel NF-κB dependent target genes that were regulated by all three subunits of the IKK complex.
3) Some NF-κB target genes were dependent on the IKKs even in the absence of extracellular stimuli, suggesting the IKK complex also regulates basal levels of NF-κB activity.
This document describes the identification of a putative human mitochondrial thymidine monophosphate kinase (TMPK2). The researchers cloned and characterized a cDNA encoding TMPK2 that contains a mitochondrial targeting sequence. They showed that TMPK2 localizes to mitochondria and overexpression increases cellular dTTP levels and the conversion of radioactive thymidine and dTMP to dTDP and dTTP in mitochondria. TMPK2 expression was detected in various tissues and cell lines, and was upregulated during monocyte/macrophage differentiation, suggesting its role in mitochondrial DNA synthesis during cellular differentiation.
The document summarizes research on the role of PARC/Cul9, an E3 ubiquitin ligase, in human embryonic stem cells. The research found that PARC/Cul9 binds to the APC7 protein, a component of the cell cycle regulating APC complex. Downregulating PARC/Cul9 increased APC7 protein levels, suggesting PARC/Cul9 negatively regulates APC7. While downregulating PARC/Cul9 did not significantly affect the overall cell cycle, it may regulate specific cell cycle phases. The research aims to further understand how PARC/Cul9 regulates stem cell self-renewal and pluripotency without inhibiting apoptosis like it does
The document summarizes a student laboratory experiment attempting to genetically transform E. coli bacterial cells with a GFP plasmid (pGLO) using heat shock. The expected results were that bacterial cells transformed with the plasmid would grow on ampicillin-containing media and glow under black light. However, the actual results found that all bacterial cells died on ampicillin-containing plates, contradicting expectations. Possible sources of error that could have caused transformation failure are discussed.
This document discusses how the transcription elongation complex (TEC), comprising the growing nascent RNA and RNA polymerase traversing chromatin, serves as a nexus for various nuclear processes including mRNA biogenesis, DNA repair, chromatin modification, and gene silencing. The TEC allows these processes to occur cotranscriptionally by localizing various protein complexes and factors to the site of transcription. In particular, the phosphorylated C-terminal domain of RNA polymerase II functions as a landing pad that recruits mRNA processing factors and chromatin modifiers to couple transcription with downstream events. This cotranscriptional coupling enhances the efficiency and coordination of gene expression and nuclear transactions organized around the transcription machinery.
Molecular Cloning of the Structural Gene for ExopolygalacturonateAlan Brooks
This document summarizes research on the cloning and characterization of a gene (pelX) from Erwinia chrysanthemi that encodes an exopolygalacturonate lyase (ExoPL). The pelX gene was cloned from a mutant strain lacking known pectate lyase genes. ExoPL was purified from a recombinant E. coli strain and characterized. A pelX mutant was constructed in E. chrysanthemi but retained pathogenicity, indicating ExoPL does not contribute to tissue maceration ability.
Transgenic mice that overexpress the Cux-1 gene in the kidney develop glomerulosclerosis and interstitial fibrosis. The study found that Cux-1 overexpression leads to increased mesangial cell proliferation and expansion of the mesangial matrix. This results in mesangial cell hyperplasia and mesangial matrix expansion. The changes are associated with disruption of podocyte architecture and loss of kidney filtration function. Overexpression of Cux-1 is sufficient to induce early changes characteristic of mesangioproliferative glomerulonephritis.
Characterization of the human HCN1 channel and its inhibition by capsazepineShahnaz Yusaf
1. The human hyperpolarization-activated cyclic nucleotide-gated 1 (hHCN1) subunit was expressed in mammalian cell lines and its electrophysiological properties were characterized using patch-clamp recordings.
2. Activation of hHCN1 generated a slowly activating, non-inactivating inward current similar to native hyperpolarization-activated currents (Ih). Ih was blocked by known blockers Cs+, ZD 7288, and zatebradine.
3. The VR1 receptor antagonist capsazepine inhibited hHCN1-mediated currents in a concentration-dependent, reversible manner by shifting the activation curve and slowing current activation kinetics.
This document summarizes a study on the heterologous expression and characterization of the c1 dioxygenase enzyme from Tetranychus urticae. Key points:
- The c1 dioxygenase gene was inserted into a plasmid and transformed into E. coli cells for expression. However, initial expression attempts did not show overexpression of the protein.
- Additional expression trials were conducted by varying culture conditions and bacterial clones. SDS-PAGE analysis showed some potential overexpression in new clones induced overnight at 37°C, though further optimization is still needed.
- Once expression is optimized, the goal is to purify the recombinant protein using its His-tag and proceed with structural characterization through crystallization,
This study aims to generate fusion-defective mutants of Chlamydomonas reinhardtii through random insertional mutagenesis using glass bead transformation. Transformed cells expressing paromomycin resistance are selected and screened for inability to mate. Several mating-deficient clones have been identified that are defective in swimming, agglutination or both. Further analysis will determine if any clones possess a fusion defect and identify the insertion location to study genes involved in fusion.
This paper describes a regulatory pathway controlling expression of Borrelia burgdorferi OspC and DbpA proteins. The study found that in B. burgdorferi strain 297, the alternative sigma factor RpoN controls expression of the alternative sigma factor RpoS. RpoS then governs expression of the outer surface lipoproteins OspC and DbpA. This regulatory network was determined through targeted gene disruption of rpoN and rpoS, followed by genetic complementation. The findings provide insight into key regulatory networks that impact B. burgdorferi pathogenesis, host range, and virulence.
This document summarizes a study investigating the oncoprotein EVI1 and its regulation of the carbonic anhydrase 3 (CA3) gene. Microarray analysis identified CA3 as a major target of EVI1. The study aims to validate changes in CA3 expression and determine the mechanism and significance of EVI1-mediated transcriptional regulation of CA3. Results show that EVI1 suppresses CA3 expression at both the mRNA and protein levels by decreasing CA3 promoter activity. This suppression of the antioxidant CA3 gene leads to increased caspase activity and apoptosis in cancer cells expressing EVI1.
1) The protein SAMP1 localizes to both the endoplasmic reticulum (ER) and microtubules, suggesting it may associate the ER with microtubules.
2) RNAi treatments to stop SAMP1 synthesis in HeLa cells resulted in changes to cell morphology, including the presence of stress fibers and abnormal mitotic cell shapes.
3) This indicates SAMP1 likely plays a role in cell adhesion and cytoplasmic structure.
- The caveolin scaffolding domain was first identified and termed in 1996. It is a 20 amino acid region within caveolin that interacts with signaling proteins like Src kinases.
- The caveolin scaffolding domain negatively regulates the activity of Src kinases and other signaling molecules by preferentially interacting with their inactive conformations.
- This scaffolding domain is important for caveolin oligomerization and organization of "pre-assembled signaling complexes" within caveolae membrane domains.
This study aims to analyze the distribution of 5-hydroxymethylcytosine (5-hmC) in the hippocampus of an Alzheimer's mouse model compared to healthy mice. DNA will be isolated from the hippocampus and analyzed using a microarray containing over 20,000 promoters and 15,000 CpG islands. Antibodies specific to 5-methylcytosine and 5-hmC will isolate DNA fragments containing these modifications, which will then be amplified and compared between the transgenic and healthy mice to assess epigenetic changes associated with Alzheimer's Disease. The results are expected to show increases, decreases, or no change in 5-hmC levels in the transgenic mouse model compared to controls.
- The study investigated the effect of calcium chloride concentration on the transformation efficiency of E. coli with plasmids pUC19 and pBR322, which differ in size.
- Maximum transformation efficiency was observed at 0.15M CaCl2 for pUC19 and 0.1M CaCl2 for the larger pBR322 plasmid.
- Increasing calcium chloride concentration above these levels decreased transformation efficiency for both plasmids, with no transformants observed above 0.2M, possibly due to decreased cell viability in hypertonic conditions.
ReedWoyda_Introducing Green Fluorescence Into Homo sapiens And Escherichia Co...Reed Woyda
This study aimed to introduce the green fluorescent protein (GFP) gene into E. coli and human cells. GFP was successfully inserted into E. coli and shown to be expressed under control of the L-arabinose promoter. Addition of restriction sites to GFP was also successful, allowing for digestion of the GFP and pcDNA plasmid. However, ligation of the digested GFP fragment into pcDNA was unsuccessful, likely due to nuclease contamination. Expression of GFP in human cells could not be verified due to a technical error during immunoblotting. While some goals were achieved, such as GFP expression in E. coli, further optimization is needed to fully introduce GFP into human cells via this methodology.
A colony to-lawn method for efficient transformation of escherichia coliCAS0609
This document describes a new method for efficiently transforming Escherichia coli called the "colony-to-lawn" method. The key steps are: 1) lysing plasmid-containing donor cells to release plasmids, 2) scraping recipient cells directly from a lawn on an agar plate into the lysate, allowing transformation without preparing competent cells. This method saves time compared to traditional methods by skipping plasmid purification and competent cell preparation. It was also used to conveniently screen positive clones after DNA ligation and transformation during construction of a mutant library.
This document discusses phospholipase C (PLC) and its role in neutrophil degranulation and T cell cytotoxicity. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. PLC signaling is required for lytic granule polarization and effective T cell killing through T cell antigen receptor activation. Neutrophil degranulation involves increases in calcium and PLC production of phosphatidylinositol, which is essential for granule exocytosis. Different intracellular signaling pathways regulate the release of primary, secondary, and tertiary granules from neutroph
This document summarizes research investigating the biosynthesis and processing of succinate dehydrogenase (SDH) subunits in cultured mammalian cells. The key points are:
1. Antisera were produced against purified bovine heart SDH and its large and small subunits, which detected precursor and mature forms of the subunits in rat, pig, and bovine cell lines.
2. In pig kidney cells, newly synthesized precursors of the large and small SDH subunits were detected that were 1000-2000 and 4000-5000 Da larger than the mature forms, respectively.
3. Pulse-chase experiments showed the precursor forms were fully processed to the mature subunits within 45 minutes when uncouplers of
The document discusses research on using phospholipase A2 (PLA2) antibodies to protect cells from radiation damage. PLA2 is an enzyme that releases fatty acids from phospholipids in cell membranes. Exposure to ionizing radiation activates PLA2, leading to cell death. The research proposal is to test whether antibodies that block PLA2 can inhibit this activation and thereby reduce the toxic effects of radiation exposure and increase radiation survival. Blocking PLA2 with antibodies may provide a new radiation protection strategy and tool for diagnosing and monitoring acute radiation sickness.
The document discusses several key topics related to DNA and its role as the genetic material:
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Similar to Taxol and 10-deacetylbaccatinIII induce distinct changes (20)
Taxol and 10-deacetylbaccatinIII induce distinct changes
1. Taxol and 10-deacetylbaccatinIII induce distinct changes
in the dynamics of caveolae
Neesar Ahmeda
, Sreekanth Dasarib
, Saumya S. Srivastavaa,1
, Amita Sneha,1
,
Absar Ahmadb
, M. Islam Khanb
, M.V. Krishnasastrya,*
a
National Centre for Cell Science, Ganeshkhind Road, University of Pune Campus, Pune 411 007, India
b
National Chemical Laboratory, Pashan Road, Pune 411 007, India
Received 27 July 2008; revised 12 September 2008; accepted 15 September 2008
Available online 24 September 2008
Edited by Lukas Huber
Abstract Taxol treatment of HeLa cells resulted in a transient
recruitment of Caveolin-1 to the cell surface followed by inter-
nalization. Interestingly, 20 min after 10-deacetylbaccatinIII
(10-DAB) treatment, the caveolae displayed faster Ôkiss and
runÕ dynamics while BaccatinIII (BacIII) did not induce any
change. Sustained phosphorylation of Caveolin-1 is observed
upon treatment and between Taxol and 10-DAB, the former
shows phosphorylated Raf-1, ERK1/2 and hyperphosphorylated
Bcl-2 while the later showed much less magnitude of the same.
BacIII treatment did not induce phosphorylation of Raf-1 or
Bcl-2. It is possible that Taxol might act on multiple targets
and the side chain may be crucial.
Ó 2008 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: Caveolae; Kiss and run dynamic; TIRFM; Taxol;
10-DeacetylbaccatinIII; BaccatinIII; Cytotoxicity
1. Introduction
Taxol has shown considerable promise as an effective anti-
tumor drug against various human cancers [1]. With the
development of the chemical method(s) for the synthesis of
the Taxol and its derivatives it became clear that it is the side
chain which apparently plays a significant role in its cytotoxic
activity [2]. Our aim of the present study was to understand the
role of the side chain of Taxol with respect to its functionality.
Hence, we compared Taxol, 10-deacetylbaccatinIII (10-DAB)
and BaccatinIII (BacIII) which differ only in the side chain.
All three drugs have been shown to induce apoptosis but might
follow apparently different pathways [3]. Recently it has been
reported that Taxol induces expression of Caveolin-1, a
marker protein of the caveolae, in MCF-7 cells and that the
Caveolin-1 phosphorylation at tyrosine-14 is necessary to en-
hance Taxol mediated cytotoxicity [4]. Considering that Taxol
is known to bind to microtubules and not caveolae per se, the
requirement and role of Caveolin-1 is not clear. Moreover,
Caveolin-1, as part of caveolae, is not an entirely static entity
as the caveolae undergo a continuous cycle of Ôkiss and runÕ
(K&R) dynamics with the plasma membrane [5]. In view of
the dynamic nature of caveolae, we examined the distribution
and change in dynamics of Caveolin-1 (or caveolae) in context
with the cytotoxicity in HeLa cells expressing GFP-Caveolin-1
by total internal reflection fluorescence microscopy (TIRFM).
2. Materials and methods
Taxol, BacIII and 10-DAB, purchased from Sigma Chemical Co.,
USA, which were dissolved in dimethylsulfoxide (DMSO) to 1 mg/ml
stock solution.
2.1. Cell culture and transfection with GFP-Caveolin-1
HeLa cells were cultured in DMEM medium supplemented with
10% (v/v) fetal calf serum and penicillin and streptomycin. All drug
treatments were done at 60–70% cell confluency. HeLa cells were trans-
fected with GFP-Caveolin-1 construct (given by Dr. A. Helenius) after
seeding them at a density of 2 · 105
cells/ml in 35 mm culture dish with
fuGENE 6 kit using 1 lg of plasmid at 6:1 reagent to plasmid ratio.
After 6 h of transfection, the medium was replaced with complete
medium.
2.2. TIRFM
The cover slip containing the HeLa cells transfected with
GFP-Caveolin-1 construct after 12 h were mounted in an Atto cham-
ber containing DMEM without phenol red and 1 mg/ml BSA. The to-
tal internal reflection angle was adjusted to observe the dynamics of
GFP-Caveolin-1 in regions of the cells in an Olympus IX-81 micro-
scope equipped with an Argon laser (488 nm line). All recordings were
performed with 100·-TIRFM (1.45NA) objective with Cascade 512B
camera at 10 Hz at 10 ms exposure time for about 90 frames for each
recording. The data were recorded before treatment and after the indi-
cated drug treatment. All the videos submitted were recorded with 25
frames per second.
2.3. Confocal microscopy
HeLa cells, after 12 h of transfection, were treated with Taxol or
BacIII or 10-DAB for a given time followed by washing with PBS.
Cells were then fixed with 3.7% paraformaldehyde (pH 7.4) in PBS,
pH 7.4, for 10 min and permeabilized with 0.1% Triton X-100 for
20 min. Non-specific binding was blocked by pretreatment with 3%
BSA in PBS for 30 min. The cells were then incubated with anti-Tubu-
lin antibody (at 1:1000 dilution) for 1 h at room temperature followed
by washing and incubation with anti-mouse Alexa-568 secondary
antibody (1:1000). The cells were then visualized in Zeiss LSM 510
Confocal microscope. Fluorescence emission was detected in 0.5 lm
optical sections.
2.4. MTT assay
To assay the viability of cell after treatment with Taxol, BacIII or
10-DAB treatment, HeLa cells were seeded at a density of 5 · 103
cells
per well in a 96-well plate and treated with various concentrations of
*
Corresponding author. Fax: +91 20 25692259.
E-mail address: mvks@nccs.res.in (M.V. Krishnasastry).
1
Authors made equal contribution.
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2008.09.029
FEBS Letters 582 (2008) 3595–3600
2. Taxol, BacIII or 10-DAB (10–0.15 lM) for 48 h in triplicates. At the
end of the treatment, the media was removed and 50 ll of MTT
(1 mg/ml) in DMEM (without phenol red) was added to each well
and incubated for another 4 h at 37 °C. Formazan crystals were solub-
lized in 50 ll of iso-propanol with shaking at room temperature for
10 min. Absorbance was measured at 570 nm using 630 nm filter.
Absorbance given by cells treated with DMSO was taken as 100%
viability.
2.5. Immunoblotting
HeLa cells (2.0 · 105
) were seeded in six well plates till they attained
70% confluency. Cells were treated with the three drugs for indicated
times. The wells were washed with PBS and the cells were scraped from
the wells to recover the cell pellet by centrifugation. The cells were then
resuspended in lysis buffer containing protease inhibitors (10 lg/ml
leupeptin, 2 mM phenylmethylsulfonylfluoride (PMSF) and 2 mM
Na3VO4). Equal amount of protein was loaded on SDS–PAGE
(10–15%) and electrophoresed proteins were transferred to a nitrocel-
lulose membrane using Tris–Glycine pH 8.0 and were probed with
anti-phosphotyrosine (PY20) (Santa Cruz, sc-508), p-Raf-1 (cell signal-
ing, #9421), p-ERK1/2 (Santa Cruz, sc-7383), ERK1/2 (Santa Cruz,
sc-94), p-P38 (Santa Cruz, sc-7973), Bcl-2 (Santa Cruz, sc-7382),
anti-mouse IgG, HRP-linked antibody (cell signaling, #7076), anti-
rabbit IgG, HRP-linked antibody (cell signaling, #7074), HRP detec-
tion reagent, LumiGLOä
reagent and peroxide (cell signaling #7003).
3. Results and discussion
The aim of the present work is to understand the role and
dynamics of Caveolin-1 in Taxol induced cytotoxicity of
mammalian cells. HeLa cells have shown both very high and
moderate expression for GFP-Caveolin-1 and for the present
work, we selected the cells that moderately expressed GFP-
Caveolin-1. Firstly, treatment with the three drugs did not in-
duce any membrane damage within 1 h of their addition as
seen by trypan blue staining of drug treated cells. The number
of viable cells remained same in comparison to untreated cells
as shown in Fig. 1A. There are several reports indicating that
Taxol, 10-DAB and BacIII induce cell death by apoptosis
[3,6–8]. The data is Fig. 1B is consistent with the published lit-
erature which shows that the HeLa cells exhibited susceptibil-
ity to all the three drugs. Different concentrations, starting
from 0.15 lM to 10 lM for 48 h, have clear cytotoxic affect
on HeLa cells. It is interesting to see that at highest concentra-
tion of Taxol (10 lM), BacIII and 10-DAB showed almost
same percentage of cell death (around 80%), as compared with
the solvent control (DMSO), but at lower concentrations
(5–0.15 lM) only Taxol was effective. Moreover, after 24 h
of treatment, only Taxol was cytotoxic to HeLa cells (data
not shown).
Caveolin-1 of caveolae is not entirely a static entity in
mammalian cell membranes as the caveolae undergo K&R
dynamics with the plasma membrane. In view of the K&R
dynamics of caveolae, we have employed total internal reflec-
tion fluorescence microscopy, which is an excellent tool for
the study of events that occur at/beneath plasma membrane.
It was interesting to see that Taxol and 10-DAB elicited dis-
tinct behavior of the GFP-Caveolin-1 dynamics [videos SV1–
SV5 and Fig. 2A (IV–IX)]. The HeLa cells expressing GFP-
Caveolin-1, before treatment, exhibited a characteristic K&R
events which are identical to the observations reported by
Pelkmans and Zerial (see video SV1 and Supplementary Video
1 in Ref. [5]). With the addition of Taxol (5 lM) there was an
immediate cell surface recruitment of GFP-Caveolin-1
(Fig. 2A-V, and video SV2). One could clearly see the forma-
tion of several new clusters of GFP-Caveolin-1 at the cell sur-
face in comparison to untreated cells (videos SV2 vs. SV1;
Fig. 2A-V vs. 2A–IV). However, after 1 h of Taxol (5 lM)
treatment there was a significant withdrawal of the GFP-Cave-
olin-1 from the cell surface (Fig. 2A-VI, and video SV3). While
no such immediate cell surface recruitment was seen in case of
10-DAB (Fig. 2A-VIII) and BacIII (Fig. 2A-XI). A very inter-
esting observation was made incase of 10-DAB (5 lM) where a
several fold increase in the radial velocity of the GFP-Caveo-
lin-1 was observed after 20 min of addition (video SV5), as
compared to the movement of the GFP-Caveolin-1 before
addition of the 10-DAB (video SV4). The motion was analyzed
by plotting the velocity histograms of Caveolae spots of several
videos. In brief, the velocity of each caveolae spot seen in vid-
eos before and after treatment was measured by obtaining the
net distance traveled by the caveolae spot divided by the time
taken to reach the end point of observation. The representative
velocity histogram, shown in Fig. 2B, highlights the normal
Fig. 1. (A) Trypanblue staining of HeLa cells post drug treatment:
Viable cells after treatment with drugs (1 h) was assayed by trypan blue
staining and the percentage of unstained cells were plotted. Data
shown are in both panels are an average of three independent
experiments. (B) Cell viability assay by MTT. HeLa cells were seeded
at a density of 5 · 103
cells per well into 96-well plate and incubated
with various concentrations of Taxol, 10-DAB and BacIII ranging
from 10 lM to 0.15 lM for 48 h and then MTT was added.
Absorbance of DMSO treated cells was taken as 100% and the viable
cells for drug treatment were plotted.
3596 N. Ahmed et al. / FEBS Letters 582 (2008) 3595–3600
3. velocity of caveolae at 3 lm/s. Interestingly, a similar calcu-
lation for 10-DAB treated caveolae shown in Fig. 2C, exhib-
ited the velocity peaked at 5 lm/s while the net percentage
of the higher velocity caveolae have gone up after treatment.
No significant change was observed either in the distribution
or in the mobility of the GFP-Caveolin-1 with DMSO alone
indicating that the dramatic mobility of 10-DAB treated cave-
olae is due to drug only (solvent control Fig. 2A, I–III). How-
ever, with BacIII (5 lM), neither an immediate cell surface
recruitment (seen in case of Taxol) nor an increase in the radial
velocity after 20 min (like 10-DAB treatment) of the GFP-
Caveolin-1 was observed but only similarity with other two
drugs was the surface withdrawal of the GFP-Caveolin-1 with-
in an hour of the treatment (Fig. 2A, X–XII). Under these con-
ditions, we have not observed any re-appearance of the
caveolae at the cell surface.
The dynamics of the GFP-Caveolin-1 by TIRFM indicate
that within an hour of the treatment with Taxol, BacIII and
10-DAB there was a significant withdrawal of the cell surface
GFP-Caveolin-1. This withdrawal may indicate an internaliza-
tion of GFP-Caveolin-1. Hence, we were interested in locating
the GFP-Caveolin-1 throughout the cell. In order to confirm
the internalization of GFP-Caveolin-1 with Taxol, 10-DAB
and BacIII treatment we analyzed z-stacks sections at an inter-
val of 0.5 lm of the different regions by Confocal microscopy
before and after drug treatment. As seen in Fig. 3, we observed
a complete internalization of GFP-Caveolin-1 after 1 h treat-
ment with these three drugs as compared to control (Control,
Fig. 3, top row) or the solvent control (DMSO, Fig. 3, second
row) which show most of the GFP-Caveolin-1 was localized at
the membrane. These Confocal studies further confirm the
internalization of the GFP-Caveolin-1 upon treatment with
these drugs.
Recent studies have shown that phosphorylation of
Caveolin-1 was essential for its internalization [9]. Since we
have seen the withdrawal of GFP-Caveolin-1 by TIRFM
Fig. 2. (A) TIRFM images of GFP-Caveolin-1 on HeLa cell basal membrane: Distribution pattern of GFP-Caveolin-1 on the cell surface of the live
HeLa cell was recorded by TIRF microscope. Still frame images from the videos showing the distributions of GFP-Caveolin-1 before and after
treatment of DMSO, Taxol, 10-DAB and BacIII (5 lM each). Videos were acquired with Cascade 512B camera for a minimum of 90 frames (4 s) or
more. Full videos have been provided as supplementary material for the panels marked with SV1–SV5. Data, shown in all panels, is one of the three
independent experiments. (B) and (C) Velocity Histograms of GFP-Caveolin-1: The motion of caveolae was analyzed by obtaining the total distance,
in pixels, moved by a caveolae vesicle in X- and Y- dimensions. Briefly, we have collected the video recordings of 90 or more consecutive frames over a
period of 3–4 s at 25 frames per second. The centroid (i.e. X- and Y- coordinates) of each caveolae spot was obtained from ImageJ software and
exported to excel spread sheet. The net distance (the sum of the distances (X1,Y 1) to (X2,Y 2) to (Xn, Y n) from frame 1–n) moved by the caveolae spot
in pixel values were calculated and converted into nanometers after taking the magnification factors of the microscope into consideration. To obtain
the velocity of a given caveolae spot, the net distance traveled by the caveolae spot was divided with the total time (in second) taken by that caveolae
frame 1–n. These velocity values are used as bin values (X-axis) and the number of caveolae that belonged to that bin value or frequency (Y-axis) were
plotted as a histogram. The symbols open circles (s) represent before 10-DAb treatment in 2B and 2 C and filled circles () after 10-DAB treatment
in 2C. For example the number of caveolae that moved less than or equal to 1000 nm is one set of bin and Y-axis points. The microscope
magnification factor is 105 nm/1 pixel. It is assumed that the caveolae spot seen in frame 2 for a given X-, Y-coordinates is same as that seen in frame
1. In view of this assumption, any spot that exhibited large mobility i.e. 4 pixels between two successive frames was not included for calculation as it
may represent noise or spurious movement.
N. Ahmed et al. / FEBS Letters 582 (2008) 3595–3600 3597
4. and cytoplasmic localization of GFP-Caveolin-1 by Confocal
microscopy we examined the phosphorylation status of GFP-
Caveolin-1. The data shown in Fig. 4A clearly shows an in-
crease in phosphorylation of Caveolin-1 after 1 h of treatment
with Taxol, 10-DAB and BacIII accompanied by an increase in
the phosphorylation of p38. The status of total tyrosine
phosphorylated proteins has not displayed any dramatic differ-
ences upon the Taxol treatment within 1 h except for the pro-
tein at 38 kDa. Moreover, Taxol treatment has resulted in an
increase in the phosphorylation of Caveolin-1 within 1 h
(Fig. 4B).
Further we were also interested in examining whether there
are any differences in the signaling pathways with these three
compounds that lead to the cell death. It has been reported
that Taxol follows the p-Raf-1, ERK pathway. Hence, we
examined the phosphorylation of Raf-1 and ERK1/2 and
found that the phosphorylation of Raf-1 and ERK1/2 in-
creases with Taxol and 10-DAB treatment but there was no
significant change in phosphorylation of the same with BacIII
(Fig. 4A) indicating of different pathways [10–13]. Moreover,
Bcl-2, which gets hyperphosphorylated within 3 h of Taxol
treatment, was also found to differ between the three drugs
in the time frame observed. The data in Fig. 4C shows an
unambiguous increase in Bcl-2 phosphorylation upon treat-
ment with Taxol (detectable after 3 h of treatment) and 10-
Fig. 3. Internalization of GFP-Caveolin-1 as examined by confocal
imaging: HeLa cells, 24 h after transfection, were treated with DMSO
(vector control), Taxol, 10-DAB and BacIII with 5 lM each for 1 h
and then processed for Confocal imaging. Blue panel show the nucleus
staining with DAPI, green panel show the distribution pattern of GFP-
Caveolin-1. In control (untreated cells), DMSO (solvent controls)
GFP-Caveolin-1 is mainly localized on the membrane but treatment
with Taxol, 10-DAB and BacIII (5 lM each) for 1 h shows significant
cytoplasmic localization. Merged images are shown in last panel. Data,
shown in all panels, is one of the three independent experiments.
Fig. 4. (A) Phosphorylation status of ERK, Raf, p38 and Caveolin-1:
Cellular protein (40 lg) per lane from HeLa cells for Control, (lane 1),
DMSO (solvent control, lane 2), Taxol 5 lM (lane 3), 10-DAB (lane 4)
and BacIII (lane 5) were treated for 1 h subjected to immunoblotting as
described materials and methods. Various panels indicate the phos-
phorylation status of kinases indicated. (B) Upper Panel): Total
phosphotyrosine content of HeLa cells upon Taxol treatment: Lane 1:
Untreated cells, Lane 2: DMSO solvent control; Lane 3: Taxol 5lM
for 5 min; Lane 4: Taxol 5 lM for 30 min; Lane 5: Taxol 5 lM for
60 min. Middle panel: Changes in the amount of phosphorylated
Caveolin-1. Bottom panel: Total Caveolin-1. (C) Phosphorylation
status of Bcl-2: Cellular protein (80 lg) per lane of HeLa cells treated
with Taxol (top panel), 10-DAB (middle) and BacIII (bottom panel)
5 lM each for 1–12 h and probed with anti-Bcl-2 antibody. Data are
one representative panel of four independent experiments.
3598 N. Ahmed et al. / FEBS Letters 582 (2008) 3595–3600
5. DAB but not with BacIII till 12 h (treatment periods exceeding
24 h may result in mild phosphorylation; see Ref. [3]).
Taxol is well known to stabilize the microtubules and also it
has a higher affinity for microtubules than 10-DAB and BacIII
[14,15,2]. It has also been shown that the Caveolin-1 move-
ment is microtubule dependent [16]. Since we have observed
a dramatic change in caveolae movement, we examined the
overall microtubule networks by Confocal microscopy. We
could not find any difference in the direct association between
GFP-Caveolin-1 and microtubules in the control and Taxol or
10-DAB or BacIII treated HeLa cells (Fig. 5A). However,
Nocodozole, which is known to inhibit polymerization of
microtubules, has increased the cell surface recruitment of
GFP-Caveolin-1. Interestingly enough, the recruitment in-
creases with time (see Fig. 5B) with little or no significant
change in the dynamics of GFP-Caveolin-1. Based on the
observations of Taxol, 10-DAB and Nocodozole treatments,
it is possible that the KR dynamics of caveolae may be
dependent on microtubule dynamics.
Considering the differences in the dynamics of GFP-Caveolin-
1, cytotoxicity and phosphorylation states of Raf-1, ERK1/2
and Bcl-2, it is clear that the observed differences are due to
the difference in the side chains, as Taxol, 10-DAB and BacIII
have the similar backbone. The KR dynamics of caveolae, in
response to EGF (70 nM), exhibit cell surface withdrawal within
10 min (see video SV6 and SV7; before EGF treatment video
SV6, after 10 min of EGF treatment video SV7) and reappear-
ance after about 40–45 min after treatment (see video SV8 and
Fig. 5C panels a–c; longer periods of observation are not possi-
ble in TIRF microscope due to movement of basal membrane
away from cover slip as a result of EGF treatment associated cell
rounding). It has been shown that the Caveolin-1 is phosphory-
lated immediately after addition of EGF and may be dephos-
phorylated (likely) as it re-appears at the cell surface. In
Fig. 5. (A) Effect of drugs on association with GFP-Caveolin-1 with microtubules: HeLa transiently expressing GFP-Caveolin-1 were plated on
coverslip and processed for Confocal imaging. Cells treated with DMSO (vector control), Taxol (5 lM), 10-DAB (5 lM) and BacIII (5 lM) for 1 h
were then fixed and labeled with anti-a-Tubulin antibody to stain microtubules and secondary antibody was Alexa-568. Blue panel show the nucleus
staining (DAPI), green panel shows the distribution pattern of GFP-Caveolin-1, red panel is the microtubule staining. Merged image is shown in the
last panel. Data are one representative panel of three independent experiments. (B) TIRF images of GFP-Caveolin-1 on HeLa cell basal membranes
of after Nocodozole treatment: Distribution pattern of GFP-Caveolin-1 at the basal cell surface of the live HeLa cells after Nocodozole treatment
recorded by TIRF microscope. Still frame images from the videos showing the distributions of GFP-Caveolin-1 before and after treatment of
Nocodozole (200 nM) at indicated times. TIRFM videos were acquired as described for Fig 2A. Data, shown in all panels, is one of the three
independent experiments. (C) TIRF images of GFP-Caveolin-1 on HeLa cell basal membranes upon EGF treatment: GFP-Caveolin-1 transfected
HeLa cells were treated with EGF (70 nM) and the videos were acquired as described for Taxol in Fig. 2. The video frame rate was 23 frames per
second. Images a–c, respectively represent GFP-Caveolin-1 before, after 10 min and 40 min treatment with EGF. Data are one representative panel
of three independent experiments. (D) TIRF images of Taxol treated Cav-1(D91-93)-GFP on HeLa cell basal membranes: Cav-1(D91–93)-GFP
transfected HeLa cells were treated with Taxol (5 lM) and the videos were acquired as described above. The video frame rate was 25 frames per
second. Panels a–c represent the images of the GFP-Caveolin-1 (D91–93) before, at 0 min (immediately after addition) and after 60 min of addition of
Taxol. Data are one representative panel of three independent experiments.
N. Ahmed et al. / FEBS Letters 582 (2008) 3595–3600 3599
6. contrast, Taxol treatment results in withdrawal and sustained
phosphorylation of Caveolin-1. Thus, it appears that this sus-
tained phosphorylation of Caveolin-1 may be necessary for the
induction of apoptosis [4]. In sharp contrast, 10-DAB induces
phosphorylation of Caveolin-1 but for apoptosis; higher con-
centrations and/or longer times are needed. We speculate that
the rapid movement (of caveolae) is likely due to non-efficient
recruitment of Raf-1 or other important molecules to the caveo-
lae as we could not detect the Raf-1 at cell membrane (same mag-
nitude that of Taxol) upon exposure of cells to 10-DAB and
BacIII (data not shown).
It is widely known that Taxol binds to Tubulin very tightly
[17]. In addition, it has been shown by molecular modeling that
the Taxol and BacIII can bind to Bcl-2 with the help of their
backbone [9]. However, there is no direct evidence of interaction
between Taxol and Caveolin-1, since Taxol, BacIII and 10-DAB
show distinct dynamics of GFP-Caveolin-1, it may be likely that
the Taxol might recognize the sites of docking of caveolae vesi-
cles (KR events) as we have consistently seen a dramatic in-
crease in the number clusters (high intensity spots in Fig. 2A,
V). It is also clear that in the absence of the side chain at car-
bon-13 of Taxol such cluster formation is completely absent.
Hence, the carbon-13 side chain of Taxol is mostly likely to
interact with the aromatic amino acids of Caveolin-1 (97
YW-
FYRL102
) which have been projected to insert into cytoplasmic
side of the cell membrane. In order to test this possibility, we
have deleted the amino acids 91–93 (91
T-F-T93
; as seen incase
of Caveolin-3 of Limb girdle muscular dystrophy-1C) of Cave-
olin-1 which has defective KR dynamics as the caveolae re-
main docked to cell membrane at large (see Fig. 5D, panel a
and video SV9: before Taxol treatment). Interestingly, upon
Taxol treatment, we could not detect cell surface recruitment
of GFP-Cav-1(D91–93)
and an immediate cluster formation of
GFP-Caveolin-1(D91–93)
(see Fig. 5D, panel b). Moreover, there
was no significant internalization of GFP-Caveolin-1(D91–93)
after 1 h of taxol (5lM) treatment either as seen from the TIRF
images (Fig. 5D, panel c and video SV10). One possible reason
for the cell surface recruitment of Caveolin-1 by Taxol could be
that the caveolae vesicles may get loaded with Taxol after
recruitment which are subsequently internalized (phosphoryla-
tion and internalization of Caveolin-1) for which the side chain
of Taxol plays an important role. To the best of our knowledge,
this is the first report describing the changes in the dynamics of
caveolae elicited by the side chains of Taxol. This model will help
us in understanding not only the significance of the side chains of
Taxol, but also the role of caveolae vesicles for the development
of resistance in certain cancers to Taxol.
Acknowledgements: The authors thank Dr. T. Yanagida and Dr. Y.
Sako for introduction to TIRFM, Dr. G. C. Mishra and Prof. A. Suro-
lia for valuable comments, and Mr. Anil Lotke and Ms. Ashwini Atre
for technical assistance. N.A, S.D., S.S. and A.S. are recipients of a se-
nior research fellowship from CSIR and UGC, Financial assistance was
provided by the Department of Biotechnology, Government of India.
Appendix A. Supplementary material
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.febslet.2008.09.029.
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