Functional conservation in the SIAMESE-RELATED family of cyclin-dependent kinase inhibitors in land plants

© 2015 American Society of Plant Biologists. All rights reserved. The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin- dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function

The plant specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis

Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA. CYCB1; 1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR

Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria

Abstract Background Cell proliferation is an important determinant of plant growth and development. In addition, modulation of cell-division rate is an important mechanism of plant plasticity and is key in adapting of plants to environmental conditions. One of the greatest challenges in understanding the cell cycle of flowering plants is the large families of CDKs and cyclins that have the potential to form many different complexes. However, it is largely unclear which complexes are active. In addition, there are many CDK- and cyclin-related proteins whose biological role is still unclear, i.e. whether they have indeed enzymatic activity. Thus, a biochemical characterization of these proteins is of key importance for the understanding of their function. Results Here we present a straightforward system to systematically express and purify active CDK-cyclin complexes from E. coli extracts. Our method relies on the concomitant production of a CDK activating kinase, which catalyzes the T-loop phosphorylation necessary for kinase activity. Taking the examples of the G1-phase cyclin CYCLIN D3;1 (CYCD3;1), the mitotic cyclin CYCLIN B1;2 (CYCB1;2) and the atypical meiotic cyclin SOLO DANCERS (SDS) in conjunction with A-, B1- and B2-type CDKs, we show that different CDKs can interact with various cyclins in vitro but only a few specific complexes have high levels of kinase activity. Conclusions Our work shows that both the cyclin as well as the CDK partner contribute to substrate specificity in plants. These findings refine the interaction networks in cell-cycle control and pinpoint to particular complexes for modulating cell proliferation activity in breeding.</p

A strategy for synthesis of lipid nanoparticles using microfluidic devices with a mixer structure

Formation behavior of lipid nanoparticles (LNPs) in microfluidic devices with a staggered herringbone micromixer (SHM) structure was investigated. The fundamental role for SHMs in LNP formation was demonstrated by determining such factors as the limiting SHM cycle numbers and the effect of flow rate. The SHM cycle numbers and the position of the first SHM were as significant as factors as the flow rate condition for producing the small-size LNPs

Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators.

Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress

Abstract 1576: Molecular profiling of the desmoplastic reaction within the colorectal tumor microenvironment using the nCounter® platform

Background &amp; Objectives: Desmoplastic reaction (DR) refers to the presence of fibrosis at the tumor's invasive margin within the tumor microenvironment (TME). DR has been associated with cancer aggressiveness across various studies in colorectal cancer (CRC). Previously, we have suggested a histological classification of DR into 3 prognostic categories; mature, intermediate or immature, based on the presence of keloid-like collagen and myxoid stroma at the extramural desmoplastic front, and we have shown that survival outcome differs significantly among the DR categories. Although DR is significantly associated with patient prognosis, its molecular background remains unclear. NanoString nCounter® analysis platform enables the multiplex gene expression analysis with up to 800 genes from a single mRNA sample. The main focus of our study is the evaluation of the molecular profiles that may drive the development of the DR types.Methods: Four CRC patients of each DR category were included in this study (n = 12). Surgical specimens were split into two halves, one of which was frozen and the other was fixed in formalin. DR was assessed on slides of the formalin-fixed tissues whereas the fresh frozen tissues samples were used for the molecular profiling. Four µm thick sections from the formalin-fixed samples were stained with hematoxylin and eosin. The extramural tumor front within the slides was assessed for DR classification. Samples containing myxoid stroma with area greater than a microscopic field of a ×40 objective lens were classified as immature. Samples with absence of size-significant myxoid stroma and presence of keloid-like collagens were classified as intermediate. Samples were classified as mature if neither size-significant myxoid stroma nor keloid-like collagens were present. Ten µm thick sections from the fresh frozen samples were stained with hematoxylin. Two regions of interest per tissue sample were selected: a) fibrotic stromal regions and b) tumor areas adjacent to the fibrotic stroma. A total area of 5mm2 was microdissected from each region of interest. RNA was extracted from each sample and over 1400 gene expressions were measured for each sample using the nCounter® PanCancer Progression and Immune Profiling panels. Nanostring nSolver™ analysis software was used for the data analysis.Results: Genes associated with cell cycle and function, epithelial to mesenchymal transition and metastasis showed significantly different expression among the stromal and tumor regions of the 3 DR categories. The distribution of immune cells within the TME was also shown to be significantly different between the DR types. Although differential gene expression was observed among all the DR classes, the immature class had the most distinct pattern of gene expression compared to the other two classes.Conclusion: This is to our knowledge the first study reporting differences in gene expression between mature, intermediate and immature DR types

TDM1 Regulation Determines the Number of Meiotic Divisions.

Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively