A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.

A-MYB (MYBL1) is a transcription factor with a role in meiosis in spermatocytes. The related B-MYB protein is a key oncogene and a master regulator activating late cell cycle genes. To activate genes, B-MYB forms a complex with MuvB and is recruited indirectly to cell cycle genes homology region (CHR) promoter sites of target genes. Activation through the B-MYB-MuvB (MMB) complex is essential for successful mitosis. Here, we discover that A-MYB has a function in transcriptional regulation of the mitotic cell cycle and can substitute for B-MYB. Knockdown experiments in cells not related to spermatogenesis show that B-MYB loss alone merely delays cell cycle progression. Only dual knockdown of B-MYB and A-MYB causes G2/M cell cycle arrest, endoreduplication, and apoptosis. A-MYB can substitute for B-MYB in binding to MuvB. The resulting A-MYB-MuvB complex activates genes through CHR sites. We find that A-MYB activates the same target genes as B-MYB. Many of the corresponding proteins are central regulators of the cell division cycle. In summary, we demonstrate that A-MYB is an activator of the mitotic cell cycle by activating late cell cycle genes.

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes.

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli.

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

☠Track: Inferred counting and tracking of replicating DNA loci.

Fluorescent microscopy is the primary method to study DNA organization within cells. However, the variability and low signal/noise commonly associated with live-cell time-lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present ★Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged, and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate ★Track's abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, ★Track can help to uncover the mechanistic principles of chromosome organization and dynamics across a range of systems.

High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS.

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB, a protein that binds to centromeric-like parS sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements of plasmid dynamics. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We develop a unified stochastic model that quantitatively explains this behaviour and predicts that cells with the lowest plasmid concentration transition to oscillatory dynamics. We confirm this prediction for F plasmid as well as a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, which according to our model, minimises ATP consumption. Our work also clarifies how various plasmid dynamics are achievable in a single unified stochastic model. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.

The importance of time of day for magnetic body alignment in songbirds.

Spontaneous magnetic alignment is the simplest known directional response to the geomagnetic field that animals perform. Magnetic alignment is not a goal directed response and its relevance in the context of orientation and navigation has received little attention. Migratory songbirds, long-standing model organisms for studying magnetosensation, have recently been reported to align their body with the geomagnetic field. To explore whether the magnetic alignment behaviour in songbirds is involved in the underlying mechanism for compass calibration, which have been suggested to occur near to sunset, we studied juvenile Eurasian reed warblers (Acrocephalus scirpaceus) captured at stopover during their first autumn migration. We kept one group of birds in local daylight conditions and an experimental group under a 2 h delayed sunset. We used an ad hoc machine learning algorithm to track the birds' body alignment over a 2-week period. Our results show that magnetic body alignment occurs prior to sunset, but shifts to a more northeast-southwest alignment afterwards. Our findings support the hypothesis that body alignment could be associated with how directional celestial and magnetic cues are integrated in the compass of migratory birds.

Center of pressure (COP) measurement in patients with confirmed successful outcomes following shoulder surgery show significant sensorimotor deficits.

PURPOSE: To determine the sensorimotor and clinical function of patients with confirmed successful outcome after either undergoing acromioclavicular joint (ACJ) stabilization, Bankart repair (BR), or rotator cuff repair (RC), and to compare these measures to the contralateral, healthy side without history of previous injuries or surgeries of the upper extremity. It was hypothesized that patients of each interventional group would have inferior sensorimotor function of the shoulder joint compared to the contralateral, healthy side, while presenting with successful clinical and functional outcomes. METHODS: Three intervention groups including ten patients who had confirmed successful clinical and functional outcomes after either undergoing ACJ stabilization, BR, or RC were evaluated postoperatively at an average follow-up of 31.7 ± 11.6 months. Additionally, a healthy control group (CG) of ten patients was included. Clinical outcomes were assessed using the Constant-Murley (CM) and American Shoulder and Elbow Surgeons (ASES) Score. Pain was evaluated using the visual analogue scale (VAS). Sensorimotor function was assessed by determining the center of pressure (COP) of the shoulder joint in a one-handed support task in supine position on a validated pressure plate. RESULTS: Each interventional group demonstrated excellent clinical outcome scores including the CM Score (ACJ 83.3 ± 11.8; BR 89.0 ± 10.3; RC 81.4 ± 8.8), ASES Score (ACJ 95.5 ± 7.0; BR 92.5 ± 9.6; RC 96.5 ± 5.2), and VAS (ACJ 0.5 ± 0.9; BR 0.5 ± 0.8; RC 0.5 ± 0.8). Overall, the CG showed no significant side-to-side difference in COP, whereas the ACJ-group and the BR-group demonstrated significantly increased COP compared to the healthy side (ACJ 103 cm vs. 98 cm, p = 0.049; BR: 116 cm vs. 102 cm, p = 0.006). The RC-group revealed no significant side-to-side difference (120 cm vs. 108 cm, n.s.). CONCLUSION: Centre of pressure measurement detected sensorimotor functional deficits following surgical treatment of the shoulder joint in patients with confirmed successful clinical and functional outcomes. This may indicate that specific postoperative training and rehabilitation protocols should be established for patients who underwent surgery of the upper extremity. These results underline that sensorimotor training should be an important component of postoperative rehabilitation and physiotherapeutic activities to improve postoperative function and joint control. LEVEL OF EVIDENCE: IV.

Ki-67 gene expression.

Ki-67 serves as a prominent cancer marker. We describe how expression of the MKI67 gene coding for Ki-67 is controlled during the cell cycle. MKI67 mRNA and Ki-67 protein are maximally expressed in G2 phase and mitosis. Expression is dependent on two CHR elements and one CDE site in the MKI67 promoter. DREAM transcriptional repressor complexes bind to both CHR sites and downregulate the expression in G0/G1 cells. Upregulation of MKI67 transcription coincides with binding of B-MYB-MuvB and FOXM1-MuvB complexes from S phase into G2/M. Importantly, binding of B-MYB to the two CHR elements correlates with loss of CHR-dependent MKI67 promoter activation in B-MYB-knockdown experiments. In knockout cell models, we find that DREAM/MuvB-dependent transcriptional control cooperates with the RB Retinoblastoma tumor suppressor. Furthermore, the p53 tumor suppressor indirectly downregulates transcription of the MKI67 gene. This repression by p53 requires p21/CDKN1A. These results are consistent with a model in which DREAM, B-MYB-MuvB, and FOXM1-MuvB together with RB cooperate in cell cycle-dependent transcription and in transcriptional repression following p53 activation. In conclusion, we present mechanisms how MKI67 gene expression followed by Ki-67 protein synthesis is controlled during the cell cycle and upon induction of DNA damage, as well as upon p53 activation.

DREAM and RB cooperate to induce gene repression and cell-cycle arrest in response to p53 activation.

Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37-/-;RB-/- cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.

Magnetic body alignment in migratory songbirds: a computer vision approach.

Several invertebrate and vertebrate species have been shown to align their body relative to the geomagnetic field. Many hypotheses have been proposed to explain the adaptive significance of magnetic body alignment outside the context of navigation. However, experimental evidence to investigate alternative hypotheses is still limited. We present a new setup to track the preferential body alignment relative to the geomagnetic field in captive animals using computer vision. We tested our method on three species of migratory songbirds and provide evidence that they align their body with the geomagnetic field. We suggest that this behaviour is involved in the underlying mechanism for compass orientation and calibration, which may occur near to sunrise and sunset periods. Our method could easily be extended to other species and used to test a large set of hypotheses to explain the mechanisms behind the magnetic body alignment and the magnetic sense in general.