Dynamics of chromatin structure and nuclear multiprotein complexes investigated by quantitative fluorescence live cell microscopy and computational modeling

Biology has rapidly been transformed into a mainly data-driven, quantitative science. Demands on biological imaging are moving towards quantitative annotations of genes in vivo. In this work I have studied in detail the spatio-temporal distribution and the molecular interaction of protein ensembles as well as of multiprotein aggregates. I have provided the methodology to estimate biophysical parameters such as diffusion coefficients, anomalous diffusion and the free fraction in the binding equilibrium of protein ensembles using fluorescence photobleaching analysis and numcerical modeling and parameter estimation. On the side of protein complexes I have extended existing single particle tracking approaches to allow to automatically detect the exact timing of mobility changes of single particles in live cells. Here, I was able to provide quantitative parameters also on the diffusion coefficient, anomalous diffusion, velocity and chromatin interaction. The nuclear protein ensemble I studied was murine linker histone H1° fused to GFP. I was able to show that diffusion and binding of H1°-GFP to chromatin can be addressed using photobleaching analysis and numcerical modeling. I have thus obtained diffusion coefficients for wild-type H1° and seven point mutants with differential binding affinity ranging from D = 0.01 mm²/s (strongest binder) to D = 0.1 mm²/s (weakest binder). Likewise, I was able to estimate the free fraction to range from = 400 ppm to = 3000 ppm. Exemplary of large multiprotein complexes I chose PML nuclear bodies (PML NBs), named after their constituent promyelotic leukemia protein. I studied in detail their dynamic mobility during early mitosis, ranging from prophase to prometaphase. A dramatic global increase in PML NB mobility was found during this period with the diffusion coefficient increasing from D = 0.001 mm²/s at interphase to D = 0.005 mm²/s at prophase. Similarly, velocities increased from v = 0.7 mm/min to v = 1.4mm/min and concomittant with a loss in subdiffusive motion. I was able to establish loss of tethering to chromatin as the most likely reason behind this increase as opposed to material flow or chromatin condensation. Lastly, I was also able to relate the timing of the mobility increase to other important cellular events. The increase of PML NB mobility predominantly occured after nuclear entry of cyclin B1, which irreversibly commits the cell to mitosis, and before nuclear envelope breakdown (NEBD)

The protein tyrosine phosphatase PPH‐7 is required for fertility and embryonic development in C. elegans at elevated temperatures

Post‐translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post‐translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH‐7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH‐7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel–Lindau (VHL) E3 ligase, suggesting a potential role for PPH‐7 in the regulation of VHL

Links across ecological scales: Plant biomass responses to elevated CO2_2

The degree to which elevated CO$_2$ concentrations (e[CO$_2$]) increase the amount of carbon (C) assimilated by vegetation plays a key role in climate change. However, due to the short-term nature of CO$_2$ enrichment experiments and the lack of reconciliation between different ecological scales, the effect of e[CO$_2$] on plant biomass stocks remains a major uncertainty in future climate projections. Here, we review the effect of e[CO$_2$] on plant biomass across multiple levels of ecological organization, scaling from physiological responses to changes in population-, community-, ecosystem-, and global-scale dynamics. We find that evidence for a sustained biomass response to e[CO$_2$] varies across ecological scales, leading to diverging conclusions about the responses of individuals, populations, communities, and ecosystems. While the distinct focus of every scale reveals new mechanisms driving biomass accumulation under e[CO$_2$], none of them provides a full picture of all relevant processes. For example, while physiological evidence suggests a possible long-term basis for increased biomass accumulation under e[CO$_2$] through sustained photosynthetic stimulation, population-scale evidence indicates that a possible e[CO$_2$]-induced increase in mortality rates might potentially outweigh the effect of increases in plant growth rates on biomass levels. Evidence at the global scale may indicate that e[CO$_2$] has contributed to increased biomass cover over recent decades, but due to the difficulty to disentangle the effect of e[CO$_2$] from a variety of climatic and land-use-related drivers of plant biomass stocks, it remains unclear whether nutrient limitations or other ecological mechanisms operating at finer scales will dampen the e[CO$_2$] effect over time. By exploring these discrepancies, we identify key research gaps in our understanding of the effect of e[CO$_2$] on plant biomass and highlight the need to integrate knowledge across scales of ecological organization so that large-scale modeling can represent the finer-scale mechanisms needed to constrain our understanding of future terrestrial C storage.ISSN:1354-1013ISSN:1365-248

Live Cell Dynamics of Promyelocytic Leukemia Nuclear Bodies upon Entry into and Exit from Mitosis

Promyelocytic leukemia nuclear bodies (PML NBs) have been proposed to be involved in tumor suppression, viral defense, DNA repair, and/or transcriptional regulation. To study the dynamics of PML NBs during mitosis, we developed several U2OS cell lines stably coexpressing PML-enhanced cyan fluorescent protein with other individual marker proteins. Using three-dimensional time-lapse live cell imaging and four-dimensional particle tracking, we quantitatively demonstrated that PML NBs exhibit a high percentage of directed movement when cells progressed from prophase to prometaphase. The timing of this increased dynamic movement occurred just before or upon nuclear entry of cyclin B1, but before nuclear envelope breakdown. Our data suggest that entry into prophase leads to a loss of tethering between regions of chromatin and PML NBs, resulting in their increased dynamics. On exit from mitosis, Sp100 and Fas death domain-associated protein (Daxx) entered the daughter nuclei after a functional nuclear membrane was reformed. However, the recruitment of these proteins to PML NBs was delayed and correlated with the timing of de novo PML NB formation. Together, these results provide insight into the dynamic changes associated with PML NBs during mitosis