Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

The formation of abnormal mitosis associated with cancer has been intriguing for many decades. While microtubules had been the focus of previous studies, recent research has focused on centrosomes, microtubule organizing centers which organize the mitotic apparatus during cell division. During normal mitosis centrosomes form two poles but in cancer, centrosomes can form three, four, or more poles, and organize tripolar, quadripolar, and multipolar mitoses, respectively. This has severe consequences for genomic stability because chromosomes are separated unequally to three, four, or more poles. This can result in aneuploidy and gene amplifications with multiple defects in cellular regulation. It can result in malignancy that is accompanied by cell cycle imbalances and abnormal cell proliferation. While radiation and chemical agents are known to damage DNA and can lead to cell cycle abnormalities, the damage of centrosome structure leading to abnormal mitosis deserves also consideration.

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Spanning the gap: unraveling RSC dynamics in vivo

Current Genetics ◽

10.1007/s00294-020-01144-1 ◽

2021 ◽

Author(s):

Heinz Neumann ◽

Bryan J. Wilkins

Keyword(s):

Cell Cycle ◽

Posttranslational Modifications ◽

Transcriptional Activation ◽

Binding Mode ◽

Binding Modes ◽

Binding Domains ◽

Binding Interface ◽

The Many ◽

And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

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TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis

Cell Death and Disease ◽

10.1038/s41419-021-03471-8 ◽

2021 ◽

Vol 12 (2) ◽

Cited By ~ 2

Author(s):

Lisenn Lalier ◽

Vincent Mignard ◽

Marie-Pierre Joalland ◽

Didier Lanoé ◽

Pierre-François Cartron ◽

...

Keyword(s):

Protein Import ◽

Glioma Cells ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Localization ◽

Antiapoptotic Protein ◽

Mitochondrial Transfer ◽

Small Domain ◽

Induction Of Apoptosis ◽

And Function

AbstractIn this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2–TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER–mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2–TOM20 interaction is thus an additional player in the control of apoptosis.

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Advanced experimental approaches to marine water-column biogeochemical processes

ICES Journal of Marine Science ◽

10.1093/icesjms/fsx146 ◽

2017 ◽

Vol 75 (1) ◽

pp. 30-42 ◽

Cited By ~ 6

Author(s):

Louis Legendre ◽

Richard B Rivkin ◽

Nianzhi Jiao

Keyword(s):

Water Column ◽

Numerical Models ◽

Free Water ◽

Sea Surface ◽

Biogeochemical Processes ◽

In Situ Experiments ◽

Technological Developments ◽

Approaches To Study ◽

Experimental Approaches

Abstract This “Food for Thought” article examines the potential uses of several novel scientific and technological developments, which are currently available or being developed, to significantly advance or supplement existing experimental approaches to study water-column biogeochemical processes (WCB-processes). After examining the complementary roles of observation, experiments and numerical models to study WCB-processes, we focus on the main experimental approaches of free-water in situ experiments, and at-sea and on-land meso- and macrocosms. We identify some of the incompletely resolved aspects of marine WCB-processes, and explore advanced experimental approaches that could be used to reduce their uncertainties. We examine three such approaches: free-water experiments of lengthened duration using bioArgo floats and gliders, at-sea mesocosms deployed several 100s m below the sea-surface using new biogeochemical sensors, and 50 m-tall on-land macrocosms. These approaches could lead to significant progress in concepts related to marine WCB-processes.

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Role of Ubiquitin Ligases and the Proteasome in Oncogenesis: Novel Targets for Anticancer Therapies

Journal of Clinical Oncology ◽

10.1200/jco.2012.44.0958 ◽

2013 ◽

Vol 31 (9) ◽

pp. 1231-1238 ◽

Cited By ~ 100

Author(s):

Lindsey N. Micel ◽

John J. Tentler ◽

Peter G. Smith ◽

Gail S. Eckhardt

Keyword(s):

Cell Cycle ◽

Anticancer Agents ◽

Cell Cycle Control ◽

Ubiquitin Proteasome System ◽

Cellular Regulation ◽

Ubiquitin Chain ◽

Key Enzyme ◽

Ubiquitin Proteasome ◽

Enzyme Families ◽

And Function

The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to cellular regulation and function. The UPS comprises a sequential series of enzymatic processes using four key enzyme families: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-carrier proteins), E3 (ubiquitin-protein ligases), and E4 (ubiquitin chain assembly factors). Because the UPS is a crucial regulator of the cell cycle, and abnormal cell-cycle control can lead to oncogenesis, aberrancies within the UPS pathway can result in a malignant cellular phenotype and thus has become an attractive target for novel anticancer agents. This article will provide an overall review of the mechanics of the UPS, describe aberrancies leading to cancer, and give an overview of current drug therapies selectively targeting the UPS.

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Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

RNA Biology ◽

10.1080/15476286.2016.1265198 ◽

2017 ◽

Vol 14 (6) ◽

pp. 726-738 ◽

Cited By ~ 34

Author(s):

Robert J. Duronio ◽

William F. Marzluff

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Histone Gene ◽

Histone Gene Expression ◽

Histone Locus Body ◽

And Function ◽

Histone Locus

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Cdc14-regulated midzone assembly controls anaphase B

The Journal of Cell Biology ◽

10.1083/jcb.200702145 ◽

2007 ◽

Vol 177 (6) ◽

pp. 981-993 ◽

Cited By ~ 101

Author(s):

Anton Khmelinskii ◽

Clare Lawrence ◽

Johanna Roostalu ◽

Elmar Schiebel

Keyword(s):

Cell Cycle ◽

Protein Phosphatase ◽

Cross Linking ◽

Aurora B ◽

Aurora B Kinase ◽

Spindle Midzone ◽

A Cell ◽

Spindle Elongation ◽

And Function ◽

Anaphase B

Spindle elongation in anaphase of mitosis is a cell cycle–regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase–Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase–Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase–Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase–Slk19 complex.

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Automatic synchronisation of the cell cycle in budding yeast through closed-loop feedback control

Nature Communications ◽

10.1038/s41467-021-22689-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Giansimone Perrino ◽

Sara Napolitano ◽

Francesca Galdi ◽

Antonella La Regina ◽

Davide Fiore ◽

...

Keyword(s):

Cell Cycle ◽

Autonomous Vehicles ◽

Genetic System ◽

Yeast Cells ◽

Control Engineering ◽

Loop Feedback ◽

Experimental Approaches ◽

Closed Loop Feedback ◽

Theoretical Foundations

AbstractThe cell cycle is the process by which eukaryotic cells replicate. Yeast cells cycle asynchronously with each cell in the population budding at a different time. Although there are several experimental approaches to synchronise cells, these usually work only in the short-term. Here, we build a cyber-genetic system to achieve long-term synchronisation of the cell population, by interfacing genetically modified yeast cells with a computer by means of microfluidics to dynamically change medium, and a microscope to estimate cell cycle phases of individual cells. The computer implements a controller algorithm to decide when, and for how long, to change the growth medium to synchronise the cell-cycle across the population. Our work builds upon solid theoretical foundations provided by Control Engineering. In addition to providing an avenue for yeast cell cycle synchronisation, our work shows that control engineering can be used to automatically steer complex biological processes towards desired behaviours similarly to what is currently done with robots and autonomous vehicles.

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Cdc6p establishes and maintains a state of replication competence during G1 phase

Journal of Cell Science ◽

10.1242/jcs.110.6.753 ◽

1997 ◽

Vol 110 (6) ◽

pp. 753-763 ◽

Cited By ~ 1

Author(s):

C.S. Detweiler ◽

J.J. Li

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Transcriptional Repression ◽

Prolonged Exposure ◽

S Phase ◽

Restrictive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Important Intermediate ◽

Initiation Of Dna Replication ◽

And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

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