Activity of the Heat Shock Protein 90 Inhibitor Ganetespib in Melanoma

Heat shock protein 90 (HSP90) is involved in the regulation of diverse biological processes such as cell signaling, proliferation and survival, and has been recently recognized as a potential target for cancer therapy. Ganetespib is a potent ATP competitive inhibitor of HSP90. Ganetespib downregulated the expression of multiple signal transducing molecules including EGFR, IGF-1R, c-Met, Akt, B-RAF and C-RAF, resulting in pronounced decrease in phosphorylation of Akt and Erk1/2 in a panel of five cutaneous melanoma cell lines including those harboring B-RAF and N-RAS mutations. Ganetespib exhibited potent antiproliferative activity on all five of these cell lines, with IC50 values between 37.5 and 84 nM. Importantly, Ganetespib is active on B-RAF mutated melanoma cells that have acquired resistance to B-RAF inhibition. Ganetespib induced apoptosis and cell cycle arrest at G1 and/or G2/M phase. Ganetespib induced cell cycle arrest was accompanied by altered expression of cyclin-dependent kinase inhibitor (CDKI) p21^Cip1 and p27^Kip1, cyclins B1, D1 and E, and/or cyclin-dependent kinases 1, 2 and 4. HSP90 is functionally important for melanoma cells and HSP90 inhibitors such as ganetespib could potentially be effective therapeutics for melanoma with various genetic mutations and acquired resistance to B-RAF inhibition.     

Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90

The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome.     

Glycogen synthase kinase 3β inhibition enhanced proliferation,migration and functional re-endothelialization of endothelial progenitor cells in hypercholesterolemia microenvironment

Hypercholesterolemia impairs the quantity and function of endothelial progenitor cell. We hypothesized that glycogen synthase kinase 3β activity is involved in regulating biological function of endothelial progenitor cells in hypercholesterolemia microenvironment. For study, endothelial progenitor cells derived from apolipoprotein E-deficient mice fed with high-fat diet were used. Glycogen synthase kinase 3β activity was interfered with glycogen synthase kinase 3β inhibitor lithium chloride or transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β (GSK3β-KM). Functions of endothelial progenitor cells, proliferation, migration, secretion and network formation of endothelial progenitor cells were assessed in vitro. The expression of phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 in endothelial progenitor cells was detected by Western blot. The in vivo function re-endothelialization and vasodilation were also analyzed by artery injury model transplanted with glycogen synthase kinase 3β-inhibited endothelial progenitor cells. We demonstrated that while the proliferation, migration, network formation as well as VEGF and NO secretion were impaired in apolipoprotein E-deficient endothelial progenitor cells, glycogen synthase kinase 3β inhibition significantly improved all these functions. Apolipoprotein E-deficient endothelial progenitor cells showed decreased phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 expression, whereas these signals were enhanced by glycogen synthase kinase 3β inhibition and accompanied with β-catenin nuclear translocation. Our in vivo model showed that glycogen synthase kinase 3β inhibition remarkably increased re-endothelial and vasodilation. Taken together, our data suggest that inhibition of glycogen synthase kinase 3β is associated with endothelial progenitor cell biological functions both in vitro and in vivo. It might be an important interference target in hypercholesterolemia microenvironment.     

Coagulansin-A has beneficial effects on the development of bovine embryos in?vitro via HSP70 induction

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 μM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 μM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 μM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 μM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro.     

UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib

HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC₅₀) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC₅₀s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC₅₀>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.Constant proteotoxic stress is a frequent occurrence in cancer cells and is derived from an adverse external microenvironment (hypoxia, acidosis) and internally from conformationally aberrant oncoproteins, high reactive oxygen species (ROS) levels, genomic instability, and stoichiometric imbalances in multi-protein machines. This stress condition raises the need for massive heat-shock chaperone support, especially from the heat-shock protein 90 (HSP90) system, to prevent protein aggregation and illicit interactions and promote tumor cell survival. Cancer-associated factors, such as mutant p53,^{1, 2} ErbB2,³ AKT,⁴ and macrophage migration inhibitory factor (MIF),^{5, 6} among others, represent HSP90 clients and require HSP90 for their stabilization in tumors. Hence, the multi-component HSP90 chaperone is highly upregulated and activated specifically in cancer cells as an adaptive response to malignancy.⁷HSP90 inhibitors have emerged as a highly promising class of anti-cancer compounds because of their ability to interfere with broadly active molecular networks, rather than a narrowly defined signaling pathway^{8, 9} and they enhance proteotoxic stress.¹⁰ Geldanamycin-based compounds represented the mainstay of HSP90 inhibition for the last 20 years.⁸ Clinically, however, these compounds proved to be of limited value due to their inherent liver and ocular toxicity coupled with only modest potency in vivo.¹¹ Major advances came with recently developed second-generation synthetic inhibitors such as the resorcinol containing compounds ganetespib (STA-9090)¹² and NVP-AUY922¹³ that are considerably more potent and less toxic. These compounds are currently being tested in phase II/III trials for their efficacy against various cancer types. Anaplastic lymphoma kinase (ALK)-driven NSCLC cancers showed particular clinical responsiveness to ganetespib.^{14, 15}Colorectal carcinoma (CRC) represents one of the most frequent malignancies worldwide, with a correspondingly high death toll. Moreover, the identification of predictive markers for patient stratification has proven to be difficult.¹⁶ HSP90 inhibition might be an attractive strategy for therapeutic improvement. However, this requires studies on how HSP90 inhibitors act on tumor cells in this cancer type.One approach to identify genes that determine cancer drug response was provided by highly parallel analysis of many cancer cell lines, specifically comparing their response with a large variety of small compounds with their gene expression patterns.^{17, 18} This strategy led to the identification of genes with expression patterns correlating with drug sensitivity. Notably, for many anticancer drugs this approach did not yield strong candidates, but in the case of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) a strong correlation was seen between drug sensitivity and the expression of functional NAD(P)H dehydrogenase quinone 1 (NQO1), an enzyme that can metabolize and activate 17-AAG.^{19, 20} Hence, correlating drug sensitivity and gene expression patterns in cell lines can identify mechanisms that determine drug response.Drugs are subjected to metabolic turnover, and a major route of excretion from the body consists in conjugation with a hydrophilic sugar moiety within the liver parenchyma, followed by secretion into the bile. A major group of enzymes that carry out such conjugations are the UDP glucuronosyltransferases (UGTs).^{21, 22, 23} These enzymes are the products of gene clusters that cover various substrate specificities. UGT substrates include bilirubin, amines, and phenol structures.²⁴ The existence of such mechanisms for drug conjugation in the liver raises the question if and under what circumstances they can be found directly in tumor cells, and presumably cause drug resistance when highly expressed.Here, we show that human CRC-derived cell lines fall into ganetespib-sensitive and -resistant groups. While the majority of CRC lines were sensitive, two lines were highly resistant. Importantly, resistant cancer cells show a high expression of the UGT1A gene, and high levels of UGT1A were shown to be critical for ganetespib turnover, drug inactivation, and cell resistance. Thus, UDP glucuronosyl conjugation detoxifies ganetespib not only in the liver but also in a subset of CRC cells, representing a potential predictive biomarker for ganetespib response in CRC and possibly other tumor types.     

HSP90 and Aha1 modulate microRNA maturation through promoting the folding of Dicer1

Aha1 is a co-chaperone of heat shock protein 90 (HSP90), and it stimulates the ATPase activity of HSP90 to promote the folding of its client proteins. By employing ascorbate peroxidase (APEX)-based proximity labeling and proteomic analysis, we identified over 30 proteins exhibiting diminished abundances in the proximity proteome of HSP90 in HEK293T cells upon genetic depletion of Aha1. Dicer1 is a top-ranked protein, and we confirmed its interactions with HSP90 and Aha1 by immunoprecipitation followed by western blot analysis. Genetic depletion of Aha1 and pharmacological inhibition of HSP90 both led to reduced levels of Dicer1 protein. Additionally, HSP90 and Aha1 bind preferentially to newly translated Dicer1. Reconstitution of Aha1-depleted cells with wild-type Aha1 substantially rescued Dicer1 protein level, and a lower level of restoration was observed for complementation with the HSP90-binding-defective Aha1-E67K, whereas an Aha1 mutant lacking the first 20 amino acids—which abolishes its chaperone activity—failed to rescue Dicer1 protein level. Moreover, knockdown of Aha1 and inhibition of HSP90 led to diminished levels of mature microRNAs (miRNAs), but not their corresponding primary miRNAs. Together, we uncovered a novel mechanism of HSP90 and Aha1 in regulating the miRNA pathway through promoting the folding of Dicer1 protein, and we also demonstrated that Aha1 modulates this process by acting as an autonomous chaperone and a co-chaperone for HSP90.     

Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM.     

Inhibition of HSP90 in Trypanosoma cruzi induces a stress response but no stage differentiation

The 90-kDa heat shock proteins (HSP90) are important in the regulation of numerous intracellular processes in eukaryotic cells. In particular, HSP90 has been shown to be involved in the control of the cellular differentiation of the protozoan parasite Leishmania donovani. We investigated the role of HSP90 in the related parasite Trypanosoma cruzi by inhibiting its function using geldanamycin (GA). GA induced a dose-dependent increase in heat shock protein levels and a dose-dependent arrest of proliferation. Epimastigotes were arrested in G₁ phase of the cell cycle, but no stage differentiation occurred. Blood form trypomastigotes showed conversion towards spheromastigote-like forms when they were cultivated with GA, but differentiation into epimastigotes was permanently blocked. We conclude that, similar to leishmanial HSP90, functional HSP90 is essential for cell division in T. cruzi and serves as a feedback inhibitor in the cellular stress response. In contrast to L. donovani cells, however, T. cruzi cells treated with GA do not begin to differentiate into relevant life cycle stages.     

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.     

siRNA-mediated knockdown against NUF2 suppresses pancreatic cancer proliferation in?vitro and in?vivo

NUF2 (NUF2, Ndc80 kinetochore complex component) plays an important role in kinetochore-microtubule attachment. It has been reported that NUF2 is associated with multiple human cancers. However, the functional role of NUF2 in pancreatic cancer remains unclear. In this study, we found that NUF2 expression was stronger in tumour tissues than in normal pancreatic tissues, and its overexpression could be related to poor prognosis. Moreover, NUF2 was highly expressed in several human pancreatic cancer cell lines. We took advantage of lentivirus-mediated siRNA (small interfering RNA) to suppress NUF2 expression in PANC-1 and Sw1990 cell lines aiming to investigate the role of NUF2 in pancreatic cancer. NUF2 silencing by RANi (RNA interference) reduced the proliferation and colony formation ability of pancreatic cancer cells in vitro. Cell cycle analysis showed that NUF2 knockdown induced cell cycle arrest at G0/G1 phase via suppression of Cyclin B1, Cdc2 and Cdc25A. More importantly, NUF2 silencing was able to alleviate in vivo tumourigenesis in pancreatic cancer xenograft nude mice. Collectively, the present study indicates that the siRNA-mediated knockdown against NUF2 may be a promising therapeutic method for the treatment of pancreatic cancer.     

Radiosynthesis,biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel ¹⁸F-labeled 5-resorcinolic triazolone derivative (1, [¹⁸F]PTP-Ganetespib) based on ganetespib. [¹⁸F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [¹⁸F]PEG-N₃ (11) with 37.3?±?5.11% of radiochemical yield and 99.7?±?0.09% of radiochemical purity. [¹⁸F]PTP-Ganetespib showed proper LogP (0.96?±?0.06) and good stability in human serum over 97% for 2?h. [¹⁸F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [¹⁸F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [¹⁸F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [¹⁸F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [¹⁸F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [¹⁸F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.     

A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.     

Long-Term Single Cell Analysis of S. pombe on a Microfluidic Microchemostat Array

Although Schyzosaccharomyces pombe is one of the principal model organisms for studying the cell cycle, surprisingly few methods have characterized S. pombe growth on the single cell level, and no methods exist capable of analyzing thousands of cells and tens of thousands of cell division events. We developed an automated microfluidic platform permitting S. pombe to be grown on-chip for several days under defined and changeable conditions. We developed an image processing pipeline to extract and quantitate several physiological parameters including cell length, time to division, and elongation rate without requiring synchronization of the culture. Over a period of 50 hours our platform analyzed over 100000 cell division events and reconstructed single cell lineages up to 10 generations in length. We characterized cell lengths and division times in a temperature shift experiment in which cells were initially grown at 30°C and transitioned to 25°C. Although cell length was identical at both temperatures at steady-state, we observed transient changes in cell length if the temperature shift took place during a critical phase of the cell cycle. We further show that cells born with normal length do divide over a wide range of cell lengths and that cell length appears to be controlled in the second generation, were large newly born cells have a tendency to divide more rapidly and thus at a normalized cell size. The platform is thus applicable to measure fine-details in cell cycle dynamics, should be a useful tool to decipher the molecular mechanism underlying size homeostasis, and will be generally applicable to study processes on the single cell level that require large numbers of precision measurements and single cell lineages.