Kimiko Tsutsui

Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

Differential expressions of the topoisomerase IIα and IIβ mRNAs in developing rat brain

Abstract Distributions of the topoisomerase IIα and IIβ mRNAs were examined in the developing rat brain, by in situ hybridization with isoform-specific oligonucleotide probes. Intense signals for the topoisomerase IIα mRNA were detected in the ventricular zone of each brain region at embryonic day 13–15 (E13–E15), and in the external granular layer of the cerebellum at postnatal day 7–14 (P7–P14). Thereafter, the signals rapidly decreased in levels and eventually disappeared from respective regions. Administration of bromodeoxyuridine (BrdU) into embryos at E13 showed that the topoisomerase IIα mRNA was expressed in the BrdU-incorporated region and its ventricular side of the neural wall, suggesting that transcription of this isoform occurs in neurons from S-phase through M-phase. On the other hand, the topoisomerase IIβ mRNA was distributed throughout the brain from E13 through P21, irrespective of the ventricular and mantle zones. Signal levels of the topoisomerase IIβ mRNA were much stronger during early developmental stages than at mature stages in various brain regions. The characteristic and differential spatio-temporal expressions suggest that the topoisomerase IIα is involved in the proliferation, while the topoisomerase IIβ is closely related to differentiation and maturation of neurons.

Immunohistochemical analyses of DNA topoisomerase II isoforms in developing rat cerebellum.

In mammalian cells, two isoforms of DNA topoisomerase II (topo IIα and topo IIβ) have been identified. Topo IIα is essential in mitotic cells, whereas the function of topo IIβ remains unclear. In the present study, we investigated the developmental control of topo II isoforms in two different neuronal lineages, cerebellar Purkinje cells and granule cells, by immunohistochemical analysis with isoform‐specific monoclonal antibodies. As expected, proliferating cells in the neuroepithelium and in the external germinal layer (EGL) were topo IIα immunopositive. The migrating as well as differentiating Purkinje cells and granule cells showed an enhanced topo IIβ immunoreactivity. The postmitotic granule cells in the postnatal EGL showed an abrupt transition of expressed topo II isoforms from IIα to IIβ. The transition was clearly coincident with the completion of final cell division and the initiation of terminal differentiation because no increase of the topo IIβ immunoreactivity was observed in the spreading EGL cells that are still in the cell division cycle. The topo IIβ signal was detected in both nucleoplasm and nucleolus of differentiating cells. However, the nucleoplasmic signal decreased significantly as the cells reached terminal differentiation. The residual topo IIβ in nucleoli was shown to occupy an unique location with respect to other nucleolar proteins, nucleolin and DNA topoisomerase I. Our findings indicate that both Purkinje cells and granule cells express the topo II isoforms in a similar timing during the cerebellar development and also suggest that topo IIβ localized in nucleoplasm is the functional entity involved in neuronal differentiation. J. Comp. Neurol. 431:228–239, 2001.

Topoisomerase IIβ Activates a Subset of Neuronal Genes that Are Repressed in AT-Rich Genomic Environment

DNA topoisomerase II (topo II) catalyzes a strand passage reaction in that one duplex is passed through a transient brake or gate in another. Completion of late stages of neuronal development depends on the presence of active β isoform (topo IIβ). The enzyme appears to aid the transcriptional induction of a limited number of genes essential for neuronal maturation. However, this selectivity and underlying molecular mechanism remains unknown. Here we show a strong correlation between the genomic location of topo IIβ action sites and the genes it regulates. These genes, termed group A1, are functionally biased towards membrane proteins with ion channel, transporter, or receptor activities. Significant proportions of them encode long transcripts and are juxtaposed to a long AT-rich intergenic region (termed LAIR). We mapped genomic sites directly targeted by topo IIβ using a functional immunoprecipitation strategy. These sites can be classified into two distinct classes with discrete local GC contents. One of the classes, termed c2, appears to involve a strand passage event between distant segments of genomic DNA. The c2 sites are concentrated both in A1 gene boundaries and the adjacent LAIR, suggesting a direct link between the action sites and the transcriptional activation. A higher-order chromatin structure associated with AT richness and gene poorness is likely to serve as a silencer of gene expression, which is abrogated by topo IIβ releasing nearby genes from repression. Positioning of these genes and their control machinery may have developed recently in vertebrate evolution to support higher functions of central nervous system.

Nuclear protein LEDGF/p75 recognizes supercoiled DNA by a novel DNA-binding domain

Lens epithelium-derived growth factor (LEDGF) or p75 is a co-activator of general transcription and also involved in insertion of human immunodeficiency virus type I (HIV-1) cDNA into host cell genome, which occurs preferentially to active transcription units. These phenomena may share an underlying molecular mechanism in common. We report here that LEDGF/p75 binds negatively supercoiled DNA selectively over unconstrained DNA. We identified a novel DNA-binding domain in the protein and termed it ‘supercoiled DNA-recognition domain’ (SRD). Recombinant protein fragments containing SRD showed a preferential binding to supercoiled DNA in vitro. SRD harbors a characteristic cluster of lysine and glutamic/aspartic acid residues. A polypeptide mimicking the cluster (K9E9K9) also showed this specificity, suggesting that the cluster is an essential element for the supercoil recognition. eGFP-tagged LEDGF/p75 expressed in the nucleus distributed partially in transcriptionally active regions that were identified by immunostaining of methylated histone H3 (H3K4me3) or incorporation of Br-UTP. This pattern of localization was observed with SRD alone but abolished if the protein lacked SRD. Thus, these results imply that LEDGF/p75 guides its binding partners, including HIV-1 integrase, to the active transcription site through recognition of negative supercoils generated around it.

The SUMO pathway is required for selective degradation of DNA topoisomerase IIβ induced by a catalytic inhibitor ICRF-1931

DNA topoisomerase I and II have been shown to be modified with a ubiquitin‐like protein SUMO in response to their specific inhibitors called ‘poisons’. These drugs also damage DNA by stabilizing the enzyme–DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIβ, but not its isoform IIα, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF‐193 which does not damage DNA. The β isoform immunoprecipitated from ICRF‐treated cells was modified by multiple modifiers, SUMO‐2/3, SUMO‐1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF‐induced degradation of topoisomerase IIβ did not occur, suggesting that the SUMO modification pathway is essential for the degradation.

Levels of topoisomerase II and DNA polymerase α are regulated independently in developing neuronal nuclei

A possible correlation between activity levels of topoisomerase II and DNA polymerase alpha was studied in neuronal nuclei from developing rat brain. A high level of canonical topoisomerase II activity was detected in neuronal nuclei throughout the development even at a late stage (28 days after birth) when the activity of DNA polymerase alpha decreased to less than 2% of the fetal level. Thus, in contrast to other systems, topoisomerase II does not change in parallel with DNA polymerase alpha during neuronal development. Our results suggest that topoisomerase II is required to maintain some fundamental processes in differentiated cells, including transcription.

Differential changes in the activities of multiple protein kinase C subspecies in the hippocampal-kindled rat

In previous studies we demonstrated that the membrane-associated protein kinase C (PKC) activities in the right and left hippocampus (HIPP) of rats kindled from the left HIPP increased significantly 4 weeks and 4 months after the last seizure compared with those in matched control rats. In this study, we investigated the long-lasting effect of HIPP-kindling on the membrane-associated activities of PKC subspecies in the bilateral HIPP 1 and 4 weeks after the last generalized kindled seizure had occurred. The membrane-associated activities of PKC subspecies were found to be subject to differential regulation. The activity of the alpha-subspecies was unchanged, whereas the respective activities of the beta- and gamma-subspecies in the kindled group increased significantly, compared with the controls, one (21%, P < 0.0001 for the beta-subspecies, and 23%, P < 0.001 for the gamma-subspecies) and 4 weeks (19%, P < 0.02 for the beta-subspecies, and 19%, P < 0.05 for the gamma-subspecies) after the last seizure. There were no significant differences in cytosolic PKC activity between the control and kindled groups for any subspecies examined at either time after the last seizure. These results suggest that activation of the PKC beta- and gamma-subspecies may play an important role in the enduring seizure susceptibility associated with kindling.

Glial reaction to photoelectric dye-based retinal prostheses implanted in the subretinal space of rats

We have designed a new type of retinal prosthesis using polyethylene films coupled with photoelectric dye molecules that absorb light and convert photon energy to electric potentials. An extruded-blown film of high-density polyethylene was used as the original polyethylene film. Recrystallized film was made by recrystallization from the melting of the original polyethylene film. A photoelectric dye,2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to the two types of polyethylene films through amide linkages. Samples of the original dye-coupled film, the dye-coupled recrystallized film, and the dye-uncoupled plain film were implanted in the subretinal space of normal adult rats. Frozen sections were cut from the eyes enucleated at 1 week or 1 month and were either stained with hematoxylin and eosin, stained immunohistochemically for glial fibrillary acidic protein (GFAP), or processed for in situ apoptosis detection. The results revealed that retinal tissue damage was negligible with no inflammatory cells and few apoptotic cells. GFAP was significantly up-regulated in retinal sites with the implantation of all types of polyethylene films at 1 week, compared with the adjacent retinal sites (P < 0.005, analysis of variance). The GFAP up-regulation was also present at 1 month for the plain film and dye-coupled recrystallized film (P < 0.05). Glial cell encirclement around the films increased significantly between 1 week and 1 month (P = 0.023, two-factor analysis of variance) but was not significantly different among the three types of polyethylene films (P = 0.4531). These results showed evidence of glial reactions to the photoelectric dye-coupled polyethylene films implanted into the subretinal space of rat eyes and also proved their basic biological safety.