Mitosis: Don't get mad, get even

The ‘mitotic spindle checkpoint’ ensures that, before a cell exits from mitosis, all of its chromosomes are aligned on the spindle to form the metaphase plate. Mad2 is an essential component of this checkpoint system and it binds specifically to unattached kinetochores.     

Genome editing in plants with MAD7 nuclease

MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1) family with a low level of homology to canonical Cas12 a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system. We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7, as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.     

Human CENP-I specifies localization of CENP-F,MAD1 and MAD2 to kinetochores and is essential for mitosis

The kinetochore, a macromolecular complex located at the centromere of chromosomes, provides essential functions for accurate chromosome segregation. Kinetochores contain checkpoint proteins that monitor attachments between the kinetochore and microtubules to ensure that cells do not exit mitosis in the presence of unaligned chromosomes. Here we report that human CENP-I, a constitutive protein of the kinetochore that shares limited similarity with Mis6 of Schizosaccharomyces pombe, is required for the localization of CENP-F and the checkpoint proteins MAD1 and MAD2 to kinetochores. Depletion of CENP-I from kinetochores causes the cell cycle to delay in G2. Although monopolar chromosomes in CENP-I-depleted cells fail to establish bipolar connections, the cells are unable to arrest in mitosis. These cells are transiently delayed in mitosis in a MAD2-dependent manner, even though their kinetochores are depleted of MAD2. The delay is extended considerably when the number of unattached kinetochores is increased. This suggests that no single unattached kinetochore in CENP-I-depleted cells can arrest mitosis. The collective output from many unattached kinetochores is required to reach a threshold signal of 'wait for anaphase' to sustain a prolonged mitotic arrest.     

Learning to get along despite struggling to get by

How cooperation can evolve by natural selection is important for understanding the evolutionary transition from unicellular to multicellular life. Here we review the evolutionary theories for cooperation, with emphasis on the mechanisms that can favor cooperation and reduce conflict in multicellular organisms.     

Functional interaction between the Arabidopsis orthologs of spindle assembly checkpoint proteins MAD1 and MAD2 and the nucleoporin NUA

In eukaryotes, the spindle assembly checkpoint (SAC) ensures the fidelity of chromosome segregation through monitoring the bipolar attachment of microtubules to kinetochores. Recently, the SAC components Mitotic Arrest Deficient 1 and 2 (MAD1 and MAD2) were found to associate with the nuclear pore complex (NPC) during interphase and to require certain nucleoporins, such as Tpr in animal cells, to properly localize to kinetochores. In plants, the SAC components MAD2, BUR1, BUB3 and Mps1 have been identified, but their connection to the nuclear pore has not been explored. Here, we show that AtMAD1 and AtMAD2 are associated with the nuclear envelope during interphase, requiring the Arabidopsis homolog of Tpr, NUA. Both NUA and AtMAD2 loss-of-function mutants have a shorter primary root and a smaller root meristem, and this defect can be partially rescued by sucrose. Mild AtMAD2 over-expressors exhibit a longer primary root, and an extended root meristem. In BY-2 cells, AtMAD2 is associated with kinetochores during prophase and prometaphase, but not metaphase, anaphase and telophase. Protein-interaction assays demonstrate binding of AtMAD2 to AtMAD1 and AtMAD1 to NUA. Together, these data suggest that NUA scaffolds AtMAD1 and AtMAD2 at the nuclear pore to form a functional complex and that both NUA and AtMAD2 suppress premature exit from cell division at the Arabidopsis root meristem.     

Trichromatic concept optimizes MAD experiments in synchrotron X-ray crystallography

The trichromatic concept is a new synchrotron beamline design that optimizes MAD experiments by reducing systematic experimental errors with three-colored and coaxial synchrotron X-ray beams produced by a tandem vertical undulator and trichromator. The concept enables rapid and flexible switching of three defined wavelengths, and extends the flexibility of experimental design for MAD data collection. Thus, we can collect MAD data taking into account time series effects such as radiation damage. The data based on the trichromatic concept gave a better quality electron density map than data collected by conventional methods. It was also revealed that multicolor diffraction using dichromatic or trichromatic X-ray beams is effective in rapid MAD data collection.     

Charge screening by cations affects the conformation of the mitochondrial inner membrane. A study of exogenous MAD(P)H oxidation in plant mitochondria.

Cations caused a decrease in the apparent Km and an increase in the Vmax. for the oxidation of exogenous NADH by both Jerusalem-artichoke (Helianthus tuberosus) and Arum maculatum (cuckoo-pint) mitochondria prepared and suspended in a low-cation medium (approximately or equal to 1 mM-K+). In Arum mitochondria the addition of cations caused a much greater stimulation of the oxidation of NAD(P)H via the cytochrome oxidase pathway than via the alternative, antimycin-insensitive, pathway. This shows that cations affected a rate-limiting step in the electron-transport chain at or beyond ubiquinone, the branch-point of electron transport in plant mitochondria. The effects were only dependent on the valency of the cation (efficiency C3+ greater than C2+ greater than C+) and not on its chemical nature, which is consistent with the theory of the diffuse layer. The results are interpreted to show that the screening of fixed negative membrane changes on lipids and protein complexes causes a conformational change in the mitochondrial inner membrane, leading to a change in a rate-limiting step of NAD(P)H oxidation. More specifically, it is proposed that screening removes electrostatic restrictions on lateral diffusion and thus accelerates diffusion-limited steps in electron transport.     

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.     

MAD contortions: conformational dimerization boosts spindle checkpoint signaling

Almost two decades after their identification, the components of the mitotic checkpoint are finally revealing their structural secrets. The activation of Mad2, a central piece of the checkpoint protein machinery, is linked to the rare ability of this protein to adopt two distinct topologies. Current models of checkpoint function propose that the topological transition between the two states of Mad2 is rate limiting for checkpoint activation and is accelerated through a self-activation process based on the direct interaction of the two Mad2 conformers. These models add a molecular framework to an old theory that depicts kinetochores as catalysts in the generation of the mitotic checkpoint signal.     

MAD2 downregulation in hypoxia is independent of promoter hypermethylation

Aberrant expression of the MAD2 protein has been linked to chromosomal instability, malignant transformation and chemoresistance. Although reduced MAD2 expression is well recognised in human cancer cell lines, the mechanism(s) underlying its down-regulation remain elusive.     

PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores

The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre–messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.