Honors & Awards
PhD Scholarship, Herchel Smith Foundation (2008-2012)
Doctor of Philosophy, University of Cambridge (2013)
Master of Science, St Petersburg Polytechnic University (2007)
Bachelor of Science, St Petersburg Polytechnic University (2005)
Jan Skotheim, Postdoctoral Faculty Sponsor
Current Research and Scholarly Interests
Cell cycle and cell size control in animal cells
Jan Skotheim, (5/20/2014)
Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.
The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in theRb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.
View details for PubMedID 30982746
A Precise Cdk Activity Threshold Determines Passage through the Restriction Point.
2018; 69 (2): 253–64.e5
At the restriction point (R), mammalian cells irreversibly commit to divide. R has been viewed as a point in G1 that is passed when growth factor signaling initiates a positive feedback loop of Cdk activity. However, recent studies have cast doubt on this model by claiming R occurs prior to positive feedback activation in G1 or even before completion of the previous cell cycle. Here we reconcile these results and show that whereas many commonly used cell lines do not exhibit a G1 R, primary fibroblasts have a G1 R that is defined by a precise Cdk activity threshold and the activation of cell-cycle-dependent transcription. A simple threshold model, based solely on Cdk activity, predicted with more than 95% accuracy whether individual cells had passed R. That a single measurement accurately predicted cell fate shows that the state of complex regulatory networks can be assessed using a few critical protein activities.
View details for PubMedID 29351845
View details for PubMedCentralID PMC5790185
Repellent and Attractant Guidance Cues Initiate Cell Migration by Distinct Rear-Driven and Front-Driven Cytoskeletal Mechanisms.
Current biology : CB
2018; 28 (6): 995–1004.e3
Attractive and repulsive cell guidance is essential for animal life and important in disease. Cell migration toward attractants dominates studies [1-8], but migration away from repellents is important in biology yet relatively little studied [5, 9, 10]. It is widely held that cells initiate migration by protrusion of their front [11-15], yet this has not been explicitly tested for cell guidance because cell margin displacement at opposite ends of the cell has not been distinguished for any cue. We argue that protrusion of the front, retraction of the rear, or both together could in principle break cell symmetry and start migration in response to guidance cues . Here, we find in the Dictyostelium model  that an attractant-cAMP-breaks symmetry by causing protrusion of the front of the cell, whereas its repellent analog-8CPT-breaks symmetry by causing retraction of the rear. Protrusion of the front of these cells in response to cAMP starts with local actin filament assembly, while the delayed retraction of the rear is independent of both myosin II polarization and of motor-based contractility. On the contrary, myosin II accumulates locally in the rear of the cell in response to 8CPT, anticipating retraction and required for it, while local actin assembly is delayed and couples to delayed protrusion at the front. These data reveal an important new concept in the understanding of cell guidance.
View details for DOI 10.1016/j.cub.2018.02.024
View details for PubMedID 29526589
View details for PubMedCentralID PMC5863766
Chemotactic Blebbing in Dictyostelium Cells.
Methods in molecular biology (Clifton, N.J.)
2016; 1407: 97-105
Many researchers use the social amoeba Dictyostelium discoideum as a model organism to study various aspects of the eukaryotic cell chemotaxis. Traditionally, Dictyostelium chemotaxis is considered to be driven mainly by branched F-actin polymerization. However, recently it has become evident that Dictyostelium, as well as many other eukaryotic cells, can also employ intracellular hydrostatic pressure to generate force for migration. This process results in the projection of hemispherical plasma membrane protrusions, called blebs, that can be controlled by chemotactic signaling.Here we describe two methods to study chemotactic blebbing in Dictyostelium cells and to analyze the intensity of the blebbing response in various strains and under different conditions. The first of these methods-the cyclic-AMP shock assay-allows one to quantify the global blebbing response of cells to a uniform chemoattractant stimulation. The second one-the under-agarose migration assay-induces directional blebbing in cells moving in a gradient of chemoattractant. In this assay, the cells can be switched from a predominantly F-actin-driven mode of motility to a bleb-driven chemotaxis, allowing one to compare the efficiency of both modes and explore the molecular machinery controlling chemotactic blebbing.
View details for DOI 10.1007/978-1-4939-3480-5_7
View details for PubMedID 27271896
Mitosis is swell.
journal of cell biology
2015; 211 (4): 733-735
Cell volume and dry mass are typically correlated. However, in this issue, Zlotek-Zlotkiewicz et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505056) and Son et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505058) use new live-cell techniques to show that entry to mitosis coincides with rapid cell swelling, which is reversed before division.
View details for DOI 10.1083/jcb.201511007
View details for PubMedID 26598610
View details for PubMedCentralID PMC4657178
How blebs and pseudopods cooperate during chemotaxis.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (32): 11703-11708
Two motors can drive extension of the leading edge of motile cells: actin polymerization and myosin-driven contraction of the cortex, producing fluid pressure and the formation of blebs. Dictyostelium cells can move with both blebs and actin-driven pseudopods at the same time, and blebs, like pseudopods, can be orientated by chemotactic gradients. Here we ask how bleb sites are selected and how the two forms of projection cooperate. We show that membrane curvature is an important, yet overlooked, factor. Dictyostelium cells were observed moving under agarose, which efficiently induces blebbing, and the dynamics of membrane deformations were analyzed. Blebs preferentially originate from negatively curved regions, generated on the flanks of either extending pseudopods or blebs themselves. This is true of cells at different developmental stages, chemotaxing to either folate or cyclic AMP and moving with both blebs and pseudopods or with blebs only. A physical model of blebbing suggests that detachment of the cell membrane is facilitated in concave areas of the cell, where membrane tension produces an outward directed force, as opposed to pulling inward in convex regions. Our findings assign a role to membrane tension in spatially coupling blebs and pseudopods, thus contributing to clustering protrusions to the cell front.
View details for DOI 10.1073/pnas.1322291111
View details for PubMedID 25074921
Bleb-driven chemotaxis of Dictyostelium cells
JOURNAL OF CELL BIOLOGY
2014; 204 (6): 1027-1044
Blebs and F-actin-driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.
View details for DOI 10.1083/jcb.201306147
View details for Web of Science ID 000333190500016
View details for PubMedID 24616222
Serum depletion of holo-ceruloplasmin induced by silver ions in vivo reduces uptake of cisplatin
JOURNAL OF INORGANIC BIOCHEMISTRY
2012; 116: 88-96
There is an emerging link between extracellular copper concentration and the uptake of cisplatin mediated by copper transporter CTR1 in cell cultures and unicellular eukaryotes. To test the link between extracellular copper level and cisplatin uptake by organs in vivo we used mice with low copper status parameters induced by AgCl-containing diet (Ag-mice). In Ag-mice, serum copper status and liver copper metabolism were characterized. It was shown that the expression level of copper transporter genes and activity of ubiquitous intracellular cuproenzymes were not affected but the level of serum holo-ceruloplasmin was not detectable. Silver was selectively absorbed by liver and accumulated in the mitochondrial matrix. Silver was present in an exchangeable form and was excreted through bile. Ag-mice model is characterized by high reproducibility, reversibility, synchronicity, and definiteness of ceruloplasmin-associated copper deficiency. After cisplatin treatment Ag-mice, as compared to control mice, demonstrated the delay in platinum uptake by organs during first 30 min. This effect was not observed at later time points probably due to cisplatin induced copper release to blood, which resulted in the recovery of copper status. These data allowed us to conclude that cisplatin uptake was coupled to copper transport in vivo.
View details for DOI 10.1016/j.jinorgbio.2012.07.003
View details for Web of Science ID 000310925700012
View details for PubMedID 23018271
[Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].
2012; 46 (2): 335-347
It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.
View details for PubMedID 22670529
Experimental switching of copper status in laboratory rodents
JOURNAL OF TRACE ELEMENTS IN MEDICINE AND BIOLOGY
2011; 25 (1): 27-35
There is an emerging link between copper metabolism, tumor growth and efficiency of antitumor treatment with platinum drugs or copper chelators. So there is an urgent need for well-defined and reproduced animal models with different states of copper metabolism. In the present study an animal model (rats and mice) with switching copper status in blood serum (copper concentration, oxidase activity and ceruloplasmin (Cp) protein content) is characterized. The drop of copper status is caused by addition of AgCl to fodder (Ag-animals). In rats and mice, the influence of silver ions on oxidase and ferroxidase activity of blood serum is similar, but copper concentration is reduced by 90% in rats, and by 60% in mice. The absorbed silver ions are transported to liver cells and included to Cp polypeptides, which are secreted to blood serum then. Cp, which circulates in bloodstream of Ag-animals contains silver atoms, and is misfolded, as judged by circular dichroism spectroscopy and differential scanning calorimetry. Single intraperitoneal or per oral injection of Cu(II) salt to Ag-animals causes recovery of oxidase and ferroxidase activity of blood serum within 4 hours in both rodent species, presumably by rapid metabolic insertion of copper into forming Cp in liver. The recovered copper status persists for 3 days under the continuing Ag-diet. The possibilities of use of Ag-rodents with switching copper status in investigation of influence of copper status on tissue-specific intracellular copper metabolism and role of copper in tumor genesis, bone metabolism and neurodegenerative diseases are discussed.
View details for DOI 10.1016/j.jtemb.2010.08.002
View details for Web of Science ID 000290884300005
View details for PubMedID 20965708
HDAC inhibitor-induced activation of NF-kappa B prevents apoptotic response of E1A+Ras-transformed cells to proapoptotic stimuli
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
2010; 42 (11): 1847-1855
HDAC inhibitors (HDACIs) are capable of suppressing the cell growth of tumour cells due to the induction of apoptosis and/or cell cycle arrest. This allows of considering HDACIs as promising agents for tumour therapy. The final outcome - apoptotic cell death or cell cycle arrest - depends on the type of tumour and cellular context. In this report, we addressed the issue by analysing effects produced in E1A+Ras-transformed MEF cells by HDAC inhibitors sodium butyrate (NaB), Trichostatin A (TSA) and some others. It has been shown that the HDACIs induced cell cycle arrest in E1A+Ras-transformed cells but not apoptosis. The antiapoptotic effect of HDACIs is likely to be a result of NF-κB-dependent signaling pathway activation. HDACI-induced activation of NF-κB takes place in spite of a deregulated PI3K/Akt pathway in E1A+Ras cells, suggesting an alternative mechanism for the activation of NF-κB based on acetylation. HDACI-dependent activation of NF-κB prevents the induction of apoptosis by cytostatic agent adriamycin and serum deprivation. Accordingly, suppression of NF-κB activity in HDACI-arrested cells by the chemical inhibitor CAPE or RelA-siRNA resulted in the induction of an apoptotic programme. Thus, our findings suggest that the activation of the NF-κB pathway in HDACI-treated E1A+Ras-transformed cells blocks apoptosis and may thereby play a role in triggering the programme of cell cycle arrest and cellular senescence.
View details for DOI 10.1016/j.biocel.2010.08.001
View details for Web of Science ID 000283698900016
View details for PubMedID 20692358
e2f1 gene is a new member of Wnt/beta-catenin/Tcf-regulated genes
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2010; 391 (1): 142-146
HDAC inhibitors induce cell cycle arrest of E1A+Ras-transformed cells accompanied by e2f1 gene down-regulation and activation of Wnt pathway. Here we show that e2f1 expression is regulated through the Wnt/Tcf-pathway: e2f1 promoter activity is inhibited by sodium butyrate (NaB) and by overexpression of beta-catenin/Tcf. The e2f1 promoter was found to contain two putative Tcf-binding elements: the proximal one competes well with canonical Tcf element in DNA-binding assay. Being inserted into luciferase reporter vector, the identified element provides positive transcriptional regulation in response to beta-catenin/Tcf co-transfection and NaB treatment. Thus we have firstly demonstrated that e2f1 belongs to genes regulated through Wnt/beta-catenin/Tcf pathway.
View details for DOI 10.1016/j.bbrc.2009.11.020
View details for Web of Science ID 000273624500026
View details for PubMedID 19900401