Saeko Takada

NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone

ABSTRACT The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein. Therefore, these two proteins, which are incorporated into the transition zone independently of each other, define different domains of the transition zone. An nphp4-null mutant forms flagella with nearly normal length, ultrastructure and intraflagellar transport. When fractions from isolated wild-type and nphp4 flagella were compared, few differences were observed between the axonemes, but the amounts of certain membrane proteins were greatly reduced in the mutant flagella, and cellular housekeeping proteins >50 kDa were no longer excluded from mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a barrier that regulates both membrane and soluble protein composition of flagella. The phenotypic consequences of NPHP4 mutations in humans likely follow from protein mislocalization due to defects in the transition zone barrier.

grp (chk1) replication-checkpoint mutations and DNA damage trigger a Chk2-dependent block at the Drosophila midblastula transition

The 13 syncytial cleavage divisions that initiate Drosophila embryogenesis are under maternal genetic control. The switch to zygotic regulation of development at the midblastula transition (MBT) follows mitosis 13, when the cleavage divisions terminate, transcription increases and the blastoderm cellularizes. Embryos mutant for grp, which encodes Checkpoint kinase 1 (Chk1), are DNA-replication-checkpoint defective and fail to cellularize, gastrulate or to initiate high-level zygotic transcription at the MBT. The mnk (also known as loki) gene encodes Checkpoint kinase 2 (Chk2), which functions in DNA-damage signal transduction. We show that mnk grp double-mutant embryos are replication-checkpoint defective but cellularize, gastrulate and activate high levels of zygotic gene expression. We also show that grp mutant embryos accumulate DNA double-strand breaks and that DNA-damaging agents induce a mnk-dependent block to cellularization and zygotic gene expression. We conclude that the DNA-replication checkpoint maintains genome integrity during the cleavage divisions, and that checkpoint mutations lead to DNA damage that induces a novel Chk2-dependent block at the MBT.

Microtubule translocation caused by three subspecies of inner-arm dynein from Chlamydomonas flagella

To help understand the function of inner‐arm dynein in nagellar motility, dynein samples from an outer arm‐missing mutant of Chlamydomonas (odal) were examined for the ability to translocate microtubules in vitro. High‐salt extract ofaxonemes containing inner‐arm dynein was separated by ion‐exchange chromatography into 7 peak fractions with ATPase activities. Of these, three fractions containing different sets of dynein heavy chains translocated microtubules. The maximal velocities were all between 3 and 5 , which were comparable to the microtubule sliding rate in disintegrating oda axonemes.

Beat Frequency Difference Between the Two Flagella of Chlamydomonas Depends on the Attachment Site of Outer Dynein Arms on the Outer-Doublet Microtubules

The two flagella of Chlamydomonas, although similar to each other at first glance, differ in functional properties. A clear difference exists in the beat frequency: the trans-flagellum (the one farthest from the eyespot) beats with 30-40% higher frequency than the cis-flagellum (the one nearest to the eyespot) in demembranated and reactivated cell models. This difference is considered to be influenced by outer arm dynein, because the two flagella beat at almost the same frequency in cell models of oda mutants lacking the outer dynein arm. When a sample of outer arm dynein extracted and purified from the wild-type axoneme was mixed with the cell models of an oda mutant, oda1, an almost normal number of outer dynein arms became attached to the axonemes, and the wild-type level of beat frequency was recovered on reactivation with ATP addition. The frequency imbalance, however, was not restored. Unexpectedly, when a similar experiment was performed with the cell model of another oda mutant, oda6, the addition of outer arm dynein restored the cis-trans frequency imbalance in addition to the normal number of outer arms and the higher level of reactivated motility. Among other oda mutants, oda3 yielded results similar to those with oda1, whereas oda2, oda4, and oda5 yielded results similar to those with oda6. Because the only structural difference between the two groups of oda mutants is that the oda1 and oda3 axonemes lack the outer arm attachment site on the outer doublet A-tubule while the axonemes of the other mutants retain it, these findings suggest that the attachment site for the outer dynein arm is important in determining the flagellar beat frequency. This suggests that the basal portion of the outer arm dynein is important in regulating the flagellar activity and therefore the behavior of the cell.

Regulation of Chk2 Ubiquitination and Signaling through Autophosphorylation of Serine 379

ABSTRACT The Chk2 protein kinase protects genome integrity by promoting cell cycle arrest or apoptosis in response to DNA double-strand breaks, and Chk2 mutations are found in both familial and sporadic cancers. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by ATM, followed by Chk2 oligomerization, auto-/transphosphorylation, and activation. Here we demonstrate that Chk2 is ubiquitinated upon activation and that this requires Chk2 kinase activity. Serine 379 (S379) was identified as a novel IR-inducible autophosphorylation site required for ubiquitination of Chk2 by a Cullin 1-containing E3 ligase complex. Importantly, S379 was required for Chk2 to induce apoptosis in cells with DNA double-strand breaks. Thus, auto-/transphosphorylation of S379 is required for Chk2 ubiquitination and effector function.

Novel TDP2-ubiquitin interactions and their importance for the repair of topoisomerase II-mediated DNA damage

Tyrosyl DNA phosphodiesterase 2 (TDP2) is a multifunctional protein implicated in DNA repair, signal transduction and transcriptional regulation. In its DNA repair role, TDP2 safeguards genome integrity by hydrolyzing 5′-tyrosyl DNA adducts formed by abortive topoisomerase II (Top2) cleavage complexes to allow error-free repair of DNA double-strand breaks, thereby conferring cellular resistance against Top2 poisons. TDP2 consists of a C-terminal catalytic domain responsible for its phosphodiesterase activity, and a functionally uncharacterized N-terminal region. Here, we demonstrate that this N-terminal region contains a ubiquitin (Ub)-associated (UBA) domain capable of binding multiple forms of Ub with distinct modes of interactions and preference for either K48- or K63-linked polyUbs over monoUb. The structure of TDP2 UBA bound to monoUb shows a canonical mode of UBA-Ub interaction. However, the absence of the highly conserved MGF motif and the presence of a fourth α-helix make TDP2 UBA distinct from other known UBAs. Mutations in the TDP2 UBA-Ub binding interface do not affect nuclear import of TDP2, but severely compromise its ability to repair Top2-mediated DNA damage, thus establishing the importance of the TDP2 UBA–Ub interaction in DNA repair. The differential binding to multiple Ub forms could be important for responding to DNA damage signals under different contexts or to support the multi-functionality of TDP2.

The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses

DNA damage induces Chk2/Mnk-dependent mitotic and developmental blocks in early Drosophila embryos. The Mnk-FHA domain and its phosphopeptide-binding ability play essential dual functions in mediating the embryonic DNA damage response: activating Mnk upon DNA damage and recruiting Mnk to multiple cellular structures independently of DNA damage.

In vitro approaches for the study of microtubule nucleation at the fission yeast spindle pole body

Publisher Summary This chapter discusses the in vitro approaches for the study of microtubule nucleation at the fission yeast spindle pole body (SPB). In Saccharomyces cerevisiae , the SPB is a disk-shaped, multilayered structure that is embedded into the nuclear envelope throughout the cell cycle. The isolation procedure requires fractionation and lysis of the nuclei to release the SPB from the nuclear envelope. These isolated preparations have been used for defining the composition and the structure of the SPB. Microtubule-nucleating activity of the budding yeast SPB has been studied, by using the partially purified SPB. This chapter describes a procedure to permeabilize S. pombe cells for studying the mechanisms of spindle elongation and of microtubule nucleation at the SPB. The mitotic SPB, but not the interphase SPB, is capable of nucleating microtubules in vitro . This result indicates that γ-tubulin localized at the SPB is in an inative form during interphase. The interphase SPB can be activated for microtubule nucleation by incubation in mitotic extracts prepared from unfertilized Xenopus eggs. This activation seems to occur through interaction of an activator present in the extracts with γ-tubulin located at the SPB. Finally, the activator has been isolated from the extracts based on the in vitro activity and identified as the ribonucleotide reductase, large subunit R1.