Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 µM dose-dependently suppressed growth, whereas LW6 at 20 µM, but not at 2-10 µM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 µM significantly promoted glucose uptake, with the strongest effect at 20 µM DIF-1, whereas LW6 at 2-20 µM significantly promoted glucose uptake, with the strongest effect at 10 µM LW6. Western blot analyses showed that LW6 (10 µM) and DIF-1 (20 µM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

Visually predicting microRNA-regulated tumor metastasis by intracellularly 3D counting of fluorescent spots based on in situ growth of DNA flares.

INTRODUCTION: MicroRNAs (miRNAs) have been revealed to be critical genetic regulators in various physiological processes and thus quantitative information on the expression level of critical miRNAs has important implications for the initiation and development of human diseases, including cancers. OBJECTIVES: We herein develop three-dimensionally (3D) counting of intracellular fluorescent spots for accurately evaluating microRNA-21 (miRNA-21) expression in individual HeLa cells based on stimuli-activated in situ growth of optical DNA flares, grid-patterned DNA-protein hybrids (GDPHs). METHODS: Target miRNA is sequence-specifically detected down to 10 pM owing to efficient signal amplification. Within living cells, GDPH flares are nuclease resistant and discrete objects with retarded mobility, enabling the screening of intracellular location and distribution of miRNAs and realizing in situ counting of target species with a high accuracy. RESULTS: The quantitative results of intracellular miRNAs by 3D fluorescence counts are consistent with qPCR gold standard assay, exhibiting the superiority over 2D counts. By screening the expression of intracellular miR-21 that can down-regulate the programmed cell death 4 (PDCD4) protein, the proliferation and migration of HeLa cells, including artificially-regulated ones, were well estimated, thus enabling the prediction of cancer metastasis in murine tumor models. CONCLUSION: The experiments in vitro, ex vivo and in vivo demonstrate that GDPH-based 3D fluorescence counts at the single cell level provide a valuable molecular tool for understanding biological function of miRNAs and especially for recognizing aggressive CTCs, offering a design blueprint for further expansion of DNA structural nanotechnology in predicting distant metastasis and prevention of tumor recurrence after primary resection.

Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Machine Learning Assisted Classification of Cell Lines and Cell States on Quantitative Phase Images.

In this report, we present implementation and validation of machine-learning classifiers for distinguishing between cell types (HeLa, A549, 3T3 cell lines) and states (live, necrosis, apoptosis) based on the analysis of optical parameters derived from cell phase images. Validation of the developed classifier shows the accuracy for distinguishing between the three cell types of about 93% and between different cell states of the same cell line of about 89%. In the field test of the developed algorithm, we demonstrate successful evaluation of the temporal dynamics of relative amounts of live, apoptotic and necrotic cells after photodynamic treatment at different doses.

Arginine-Arginine-Leucine Peptide Targeting Heat Shock Protein 70 for Cancer Imaging.

Arg-Arg-Leu (RRL) is a potent tumor-homing tripeptide. However, the binding target is unclear. In this study, we intended to identify the binding target of RRL and evaluate the tumor targeting of 99mTc-MAG3-RRL in vivo. Biotin-RRL, 5-TAMRA-RRL, and 99mTc-MAG3-RRL were designed to trace the binding target and tumor lesion. Immunoprecipitation-mass spectrometry was conducted to identify the candidate proteins and determination of the subcellular localization was also performed. A pull-down assay was performed to demonstrate the immunoprecipitate. Fluorescence colocalization and cell uptake assays were performed to elucidate the correlation between the selected binding protein and RRL, and the internalization mechanism of RRL. Biodistribution and in vivo imaging were performed to evaluate the tumor accumulation and targeting of 99mTc-MAG3-RRL. The target for RRL was screened to be heat shock protein 70 (HSP70). The prominent uptake distribution of RRL was concentrated in the membrane and cytoplasm. A pull-down assay demonstrated the existence of HSP70 in the biotin-RRL captured complex. Regarding fluorescence colocalization and cell uptake assays, RRL may interact with HSP70 at the nucleotide-binding domain (NBD). Clathrin-dependent endocytosis and macropinocytosis could be a vital internalization mechanism of RRL. In vivo imaging and biodistribution both demonstrated that 99mTc-MAG3-RRL can trace tumors with satisfactory accumulation in hepatoma xenograft mice. The radioactive signals accumulated in tumor lesions can be blocked by VER-155008, which can bind to the NBD of HSP70. Our findings revealed that RRL may interact with HSP70 and that 99mTc-MAG3-RRL could be a prospective probe for visualizing overexpressed HSP70 tumor sections.

Golgi stress response: A regulatory mechanism of Golgi function.

The organelle of eukaryotes is a finely regulated system. Once disturbed, it activates the specific autoregulatory systems, namely, organelle autoregulation. Among which, the Golgi stress response accounts for one. When the abundance and capacity of the Golgi apparatus are insufficient compared with cellular demand, the Golgi stress response is activated to enhance the function of the Golgi apparatus. Although the molecular mechanism of the Golgi stress response has not been well characterized yet, it seems to be an important part of the mammalian stress response. In this review, we discuss the current status of research on the six pathways of the mammalian Golgi stress response (the TFE3, heat shock protein 47, CREB3, E26 transformation specific, proteoglycan, and mucin pathways), which regulate the general function of the Golgi apparatus, anti-apoptosis, pro-apoptosis, proteoglycan glycosylation, and mucin glycosylation, respectively.

Optogenetic Control of Non-Apoptotic Cell Death.

Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.

CHIMs are versatile cholesterol analogs mimicking and visualizing cholesterol behavior in lipid bilayers and cells.

Cholesterol is an essential component of cellular membranes regulating the structural integrity and fluidity of biological bilayers and cellular processes such as signal transduction and membrane trafficking. However, tools to investigate the role and dynamics of cholesterol in live cells are still scarce and often show limited applicability. To address this, we previously developed a class of imidazolium-based cholesterol analogs, CHIMs. Here we confirm that CHIM membrane integration characteristics largely mimic those of cholesterol. Computational studies in simulated phospholipid bilayers and biophysical analyses of model membranes reveal that in biologically relevant systems CHIMs behave similarly to natural cholesterol. Importantly, the analogs can functionally replace cholesterol in membranes, can be readily labeled by click chemistry and follow trafficking pathways of cholesterol in live cells. Thus, CHIMs represent chemically versatile cholesterol analogs that can serve as a flexible toolbox to study cholesterol behavior and function in live cells and organisms.

Homeostasis of the ER redox state subsequent to proteasome inhibition.

Endoplasmic reticulum (ER) maintains within, an oxidative redox state suitable for disulfide bond formation. We monitored the ER redox dynamics subsequent to proteasome inhibition using an ER redox probe ERroGFP S4. Proteasomal inhibition initially led to oxidation of the ER, but gradually the normal redox state was recovered that further led to a reductive state. These events were found to be concomitant with the increase in the both oxidized and reduced glutathione in the microsomal fraction, with a decrease of total intracellular glutathione. The ER reduction was suppressed by pretreatment of a glutathione synthesis inhibitor or by knockdown of ATF4, which induces glutathione-related genes. These results suggested cellular adaptation of ER redox homeostasis: (1) inhibition of proteasome led to accumulation of misfolded proteins and oxidative state in the ER, and (2) the oxidative ER was then reduced by ATF4 activation, followed by influx of glutathione into the ER.

S100A16 Regulates HeLa Cell through the Phosphatidylinositol 3 Kinase (PI3K)/AKT Signaling Pathway.

BACKGROUND S100 calcium-binding protein A16 (S100A16) is closely related to the onset and progression of tumors. MATERIAL AND METHODS In the research, the mainly purpose was to investigate the effect of S100A16 on the proliferation ability, invasion, and angiogenesis of HeLa cells. An adenoviral vector overexpressing S100A16 (Ad-S100A16) was constructed and transfected into HeLa cells, forming a stable cells line of overexpression. The effect of S100A16 on the proliferative capacity of HeLa cells was evaluated by a Cell Counting Kit-8 (CCK-8) assay. Cell migration capacity was determined by a Transwell migration assay. Changes in matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, and vimentin expression were evaluated by a cell-based immunofluorescence assay. The effect of S100A16 on angiogenesis was verified by knockout experiment. RESULTS Overexpression of S100A16 significantly enhanced the proliferative and migratory capacities of HeLa cells (P<0.05), upregulated expression of matrix MMP-2, MMP-9, vimentin, phosphatidylinositol 3 kinase, and phosphorylated protein kinase B, and downregulated expression of E-cadherin. Vascular endothelial growth factor expression increased, phosphatase and tensin homolog expression decreased, and angiogenesis was positively correlated with S100A16 expression. These effects were largely mediated by the activation of the phosphatidylinositol 3 kinase/protein kinase B pathways. CONCLUSIONS S100A16 could promote the proliferation, migration, and tumor angiogenesis of HeLa cells by regulating the phosphatidylinositol 3 kinase/protein kinase B signaling pathways.

The Invasion Plasmid Antigen J (IpaJ) from Salmonella Inhibits NF-κB Activation by Suppressing IκBα Ubiquitination.

Salmonella enterica serovar Pullorum is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. In contrast to the strong inflammatory response induced by Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, S Pullorum causes systemic infection with little inflammation. The effector proteins secreted by Salmonella often play a crucial role in modulating host signal transduction and cellular processes to the pathogen's advantage. In the present study, the invasion plasmid antigen J (IpaJ) protein specifically identified in S Pullorum was found to significantly inhibit activation of the key proinflammatory transcription factor, NF-κB, which was induced by tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and lipopolysaccharide (LPS). IpaJ inhibited the NF-κB pathway in cells infected with S Pullorum through the stabilization of IκBα. Deletion of ipaJ in S Pullorum caused a significantly increased level of ubiquitinated IκBα that was subsequently degraded by the proteasome in HeLa cells. Moreover, IpaJ was efficient in the prevention of NF-κB translocation to the nucleus and ultimately interfered with the secretion of the proinflammatory cytokines IL-1ß, IL-6, and IL-8 in infected HeLa cells. Additionally, the transformation of ipaJ into S Enteritidis decreased the secretion of proinflammatory cytokines in HeLa cells through suppression of the NF-κB pathway. The infection of chicken peripheral blood monocyte-derived macrophages (chMDM) confirmed that ipaJ-deleted S Pullorum induced a stronger expression of proinflammatory cytokines than the wild-type and complementary strains. In summary, the present study revealed that IpaJ functions as an important anti-inflammatory protein involved in S Pullorum infection through inhibition of the NF-κB pathway and the subsequent inflammatory response.

Glycolytic suppression dramatically changes the intracellular metabolic profile of multiple cancer cell lines in a mitochondrial metabolism-dependent manner.

Most cancer cells rely on glycolysis to generate ATP, even when oxygen is available. However, merely inhibiting the glycolysis is insufficient for the eradication of cancer cells. One main reason for this is that cancer cells have the potential to adapt their metabolism to their environmental conditions. In this study, we investigated how cancer cells modify their intracellular metabolism when glycolysis is suppressed, using PANC-1 pancreatic cancer cells and two other solid tumor cell lines, A549 and HeLa. Our study revealed that glycolytically suppressed cells upregulated mitochondrial function and relied on oxidative phosphorylation (OXPHOS) to obtain the ATP necessary for their survival. Dynamic changes in intracellular metabolic profiles were also observed, reflected by the reduced levels of TCA cycle intermediates and elevated levels of most amino acids. Glutamine and glutamate were important for this metabolic reprogramming, as these were largely consumed by influx into the TCA cycle when the glycolytic pathway was suppressed. During the reprogramming process, activated autophagy was involved in modulating mitochondrial function. We conclude that upon glycolytic suppression in multiple types of tumor cells, intracellular energy metabolism is reprogrammed toward mitochondrial OXPHOS in an autophagy-dependent manner to ensure cellular survival.

Novel miR-29b target regulation patterns are revealed in two different cell lines.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b, have been detected in various biological fluids and tissues from a large variety of disease models. However, how miRNAs "react" and function in different cellular environments is still largely unknown. In this study, the regulation patterns of miR-29b between human and mouse cell lines were compared for the first time. CRISPR/Cas9 gene editing was used to stably knockdown miR-29b in human cancer HeLa cells and mouse fibroblast NIH/3T3 cells with minimum off-targets. Genome editing revealed mir-29b-1, other than mir-29b-2, to be the main source of generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells.

Site-specific N-glycosylation of HeLa cell glycoproteins.

We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media - nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.

TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages.

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-ß. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.

Doxorubicin-Induced Cancer Cell Senescence Shows a Time Delay Effect and Is Inhibited by Epithelial-Mesenchymal Transition (EMT).

BACKGROUND Senescence is a natural barrier for the body to resist the malignant transformation of its own cells. This work investigated the senescence characteristics of cancer cells in vitro. MATERIAL AND METHODS Human cervical cancer HeLa cells were treated with different concentrations of doxorubicin for 3 days, with or without subsequent extended culture in drug-free medium for 6 days. Senescent cell ratios between these 2 culture schemes were calculated. Expression of 2 senescence-associated secretory factors, IL-6 and IL-8, were detected by RT-PCR and ELISA. Doxorubicin treatment induced epithelial-mesenchymal transition in cancer cells. The proportions of senescent cells in epithelial-like and mesenchymal-like sub-groups were calculated. Doxorubicin-treated HeLa cells were stained with Vimentin antibody and sorted by flow cytometry. Senescent cell marker p16ᴵᴺᴷ4ᵃ and IL-8 expression in Vimentin-high and Vimentin-low cells were detected by Western blot. RESULTS We found that less than 1% of HeLa cells showed senescence phenotype after treatment with doxorubicin for 3 days. However, the proportion of senescent cells was significantly increased when the doxorubicin-treated cells were subsequently cultured in drug-free medium for another 6d. RT-PCR and ELISA results showed that this prolonged culture method could further improve the expression of IL-6 and IL-8. We also found that the senescent cells were mainly epithelial-like type and few presented mesenchymal-like shape. p16ᴵᴺᴷ4ᵃ and IL-8 expression were decreased in cell fraction with higher Vimentin expression. CONCLUSIONS Our results suggested the existence of time delay effect in doxorubicin-induced senescence of HeLa cells, and epithelial- mesenchymal transition may resist doxorubicin-induced cell senescence.

Modeling studies of heterogeneities in glycolytic oscillations in HeLa cervical cancer cells.

Previous experiments demonstrated that a population of HeLa cells starved of glucose or both glucose and serum exhibited a strong heterogeneity in the glycolytic oscillations in terms of the number of oscillatory cells, periods of oscillations, and duration of oscillations. Here, we report numerical simulations of this heterogeneous oscillatory behavior in HeLa cells by using a newly developed mathematical model. It is simple enough that we can apply a mathematical analysis, but capture the core of the glycolytic pathway and the activity of the glucose transporter (GLUT). Lognormal distributions of the values of the four rate constants in the model were obtained from the experimental distributions in the periods of oscillations. Thus, the heterogeneity in the periods of oscillations can be attributed to the difference in the rate constants of the enzymatic reactions. The activity of GLUT is found to determine whether the HeLa cells were oscillatory or non-oscillatory under the same experimental conditions. Simulation with the log-normal distribution of the maximum uptake velocity of glucose and the four randomized rate constants based on the log-normal distributions successfully reproduced the time-dependent number of oscillatory cells (oscillatory ratios) under the two starving conditions. The difference in the initial values of the metabolites has little effect on the simulated results.

GRASP depletion-mediated Golgi destruction decreases cell adhesion and migration via the reduction of α5ß1 integrin.

The Golgi apparatus is a membrane-bound organelle that serves as the center for trafficking and processing of proteins and lipids. To perform these functions, the Golgi forms a multilayer stacked structure held by GRASP55 and GRASP65 trans-oligomers and perhaps their binding partners. Depletion of GRASP proteins disrupts Golgi stack formation and impairs critical functions of the Golgi, such as accurate protein glycosylation and sorting. However, how Golgi destruction affects other cellular activities is so far unknown. Here, we report that depletion of GRASP proteins reduces cell attachment and migration. Interestingly, GRASP depletion reduces the protein level of α5ß1 integrin, the major cell adhesion molecule at the surface of HeLa and MDA-MB-231 cells, due to decreased integrin protein synthesis. GRASP depletion also increases cell growth and total protein synthesis. These new findings enrich our understanding on the role of the Golgi in cell physiology and provide a potential target for treating protein-trafficking disorders.

A new benzofuran from Artemisia halodendron Turcz. ex Bess.

A new benzofuran, methyl (2S,2″S,3'E)-[2-(1″-acetoxypropan-2-yl)-2,3-dihydrobenzofuran-5-yl]acrylate (1), and 13 known compounds (2-14) were isolated from an ethanol extract of Artemisia halodendron Turcz. ex Bess. The chemical structures of these compounds were determined by 1D and 2D NMR (1H-1H COSY, HMBC, HMQC and NOESY) and HR-ESI-MS spectra, and results were compared with data from the literature. The effects of compounds 1-14 were measured on NF-κB activation, with compounds 2 and 3 exhibiting inhibitory activities against TNF-α-induced NF-κB reporter gene expression in HeLa cells from 10 to 100 µM.

Albatross/FBF1 contributes to both centriole duplication and centrosome separation.

The centrosome is a small but important organelle that participates in centriole duplication, spindle formation, and ciliogenesis. Each event is regulated by key enzymatic reactions, but how these processes are integrated remains unknown. Recent studies have reported that ciliogenesis is controlled by distal appendage proteins such as FBF1, also known as Albatross. However, the precise role of Albatross in the centrosome cycle, including centriole duplication and centrosome separation, remains to be determined. Here, we report a novel function for Albatross at the proximal ends of centrioles. Using Albatross monospecific antibodies, full-length constructs, and siRNAs for rescue experiments, we found that Albatross mediates centriole duplication by recruiting HsSAS-6, a cartwheel protein of centrioles. Moreover, Albatross participates in centrosome separation during mitosis by recruiting Plk1 to residue S348 of Albatross after its phosphorylation. Taken together, our results show that Albatross is a novel protein that spatiotemporally integrates different aspects of centrosome function, namely ciliogenesis, centriole duplication, and centrosome separation.