Assembly,molecular organization,and membrane-binding properties of development-specific septins

Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28–Spr3–Cdc3–Cdc10–Cdc10–Cdc3–Spr3–Spr28 hetero-octamer. Only coexpressed Spr3 and Spr28 colocalize with Cdc3 and Cdc10 in mitotic cells, indicating that incorporation requires a Spr28-Spr3 protomer. Unlike their mitotic counterparts, Spr28-Spr3–capped rods are unable to form higher-order structures in solution but assemble to form long paired filaments on lipid monolayers containing phosphatidylinositol-4,5-bisphosphate, mimicking presence of this phosphoinositide in the prospore membrane. Spr28 and Spr3 fail to rescue the lethality of a cdc11Δ cdc12Δ mutant, and Cdc11 and Cdc12 fail to restore sporulation proficiency to spr3Δ/spr3Δ spr28Δ/spr28Δ diploids. Thus, specific meiotic and mitotic subunits endow septin complexes with functionally distinct properties.     

Intraocular iron injection induces oxidative stress followed by elements of geographic atrophy and sympathetic ophthalmia

Iron has been implicated in the pathogenesis of age‐related retinal diseases, including age‐related macular degeneration (AMD). Previous work showed that intravitreal (IVT) injection of iron induces acute photoreceptor death, lipid peroxidation, and autofluorescence (AF). Herein, we extend this work, finding surprising chronic features of the model: geographic atrophy and sympathetic ophthalmia. We provide new mechanistic insights derived from focal AF in the photoreceptors, quantification of bisretinoids, and localization of carboxyethyl pyrrole, an oxidized adduct of docosahexaenoic acid associated with AMD. In mice given IVT ferric ammonium citrate (FAC), RPE died in patches that slowly expanded at their borders, like human geographic atrophy. There was green AF in the photoreceptor ellipsoid, a mitochondria‐rich region, 4 h after injection, followed later by gold AF in rod outer segments, RPE and subretinal myeloid cells. The green AF signature is consistent with flavin adenine dinucleotide, while measured increases in the bisretinoid all‐trans‐retinal dimer are consistent with the gold AF. FAC induced formation carboxyethyl pyrrole accumulation first in photoreceptors, then in RPE and myeloid cells. Quantitative PCR on neural retina and RPE indicated antioxidant upregulation and inflammation. Unexpectedly, reminiscent of sympathetic ophthalmia, autofluorescent myeloid cells containing abundant iron infiltrated the saline‐injected fellow eyes only if the contralateral eye had received IVT FAC. These findings provide mechanistic insights into the potential toxicity caused by AMD‐associated retinal iron accumulation. The mouse model will be useful for testing antioxidants, iron chelators, ferroptosis inhibitors, anti‐inflammatory medications, and choroidal neovascularization inhibitors.     

Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.     

The Transition of Closely Opposed Lesions to Double-Strand Breaks during Long-Patch Base Excision Repair Is Prevented by the Coordinated Action of DNA Polymerase δ and Rad27/Fen1

DNA double-strand breaks can result from closely opposed breaks induced directly in complementary strands. Alternatively, double-strand breaks could be generated during repair of clustered damage, where the repair of closely opposed lesions has to be well coordinated. Using single and multiple mutants of Saccharomyces cerevisiae (budding yeast) that impede the interaction of DNA polymerase δ and the 5′-flap endonuclease Rad27/Fen1 with the PCNA sliding clamp, we show that the lack of coordination between these components during long-patch base excision repair of alkylation damage can result in many double-strand breaks within the chromosomes of nondividing haploid cells. This contrasts with the efficient repair of nonclustered methyl methanesulfonate-induced lesions, as measured by quantitative PCR and S1 nuclease cleavage of single-strand break sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than does widely spaced damage in order to prevent the development of double-strand breaks.Endogenous metabolism or environmental factors such as oxidizing and alkylating agents can produce a wide variety of lesions in DNA. The genomes of mammalian cells experience from 10,000 to as many as 200,000 modifications per day (37, 44). Most lesions are repaired by a complex network of proteins that are part of an elaborate, multistep base excision repair (BER) system that generates single-strand break (SSB) intermediates. Importantly, defects in BER can lead to malignancies and can be associated with age-associated disease, especially neurodegeneration (60).BER is initiated by specific DNA N-glycosylases that remove damaged bases, yielding apurinic/apyrimidinic (AP) sites. Subsequent incision by AP endonucleases results in SSBs, and excision results in a single base gap as a repair intermediate (33, 53). SSBs are expected to be frequent in the genome due to the abundance of base damage as well as intermediates of repair, recombination, replication, and other DNA transactions (15, 16). Because they are generally repaired efficiently by BER and SSB repair enzymes (16, 57), SSBs per se may not be a major source of genome instability. However, if lesions are clustered, the formation of two closely spaced SSBs on opposing strands (or a single SSB and a modified nucleotide or AP site) might pose a special risk in terms of the potential to generate mutations or the possibility of conversion to double-strand breaks (DSBs), which are potent genotoxic lesions. Clustered lesions can arise within cells by chance association of random DNA lesions in a small region or the induction of multiple events in a narrow region, as found for ionizing radiation and various chemicals, such as those used in cancer treatments (47, 58, 59). While efficient BER is important for genome integrity, the repair must be well coordinated to avoid the generation of closely opposed SSB intermediates at closely spaced lesions that could result in the secondary generation of DSBs, especially since cells have limited DSB repair capacity (<50 DSBs/cell in the case of Saccharomyces cerevisiae) (48). While the impact of clustered lesions on repair of DNA has been examined in vitro by use of purified enzymes or cell extracts (13, 14, 27, 39, 56), there has been little opportunity to address specifically the repair of clustered lesions, except for those arising from UV damage (49).Whether formed directly from sugar damage or as BER intermediates, SSBs formed during the repair of base damage often possess 5′-deoxyribose phosphate (5′-dRP) ends that are not suitable for rejoining by DNA ligases (9, 15). In humans, removal and repair of 5′-dRP are accomplished by different combinations of proteins (3, 15) that result in short-patch repair, involving replacement of a single nucleotide (nt), or long-patch repair, involving 2 to 10 nt. The budding yeast Saccharomyces cerevisiae lacks a DNA polymerase β that provides AP lyase activity required for short-patch repair in mammalian cells. Instead, removal and repair of a 5′-dRP rely on the long-patch pathway, involving the successive actions of DNA polymerase δ (Pol δ) for strand displacement, the Rad27/Fen1 endonuclease to remove 5′ flaps, and DNA ligase (Cdc9) to rejoin the resulting nicks (9). The sliding clamp protein PCNA, which interacts with all three players, has been proposed to play a central role in coordinating these processes (18, 19, 34). The coupling between the strand displacement reaction by Pol δ and the flap cutting reaction by Fen1 is highly efficient, with over 90% of the products released by Fen1 being mononucleotides (17).Although the coordination of Pol δ, PCNA, and Rad27/Fen1 provides efficient processing of individual lesions in DNA, closely opposed SSBs that arise during repair of base damage could manifest as DSBs, either directly or as a result of SSB processing. A DNA damaging agent that has been used frequently to characterize long- and short-patch BER is methyl methanesulfonate (MMS). Recently, we described the detection of closely opposed MMS-induced lesions in yeast (42). Since the closely opposed lesions might represent a special challenge to BER, we considered the possibility that they might specifically impact long-patch repair through Pol δ and/or coordination of events with Rad27/Fen1. Pol δ of S. cerevisiae is a heterotrimeric enzyme consisting of Pol3, Pol31, and Pol32 (23). The nonessential Pol32 subunit is involved in translesion DNA synthesis (TLS) (24, 30) and also break-induced replication (41). However, its role in other types of DNA repair remains unclear. Using our in vivo assay for specifically detecting closely spaced methylated DNA lesions (42) and SSBs, we examined the role of Pol32 as well as the cooperation between Pol δ, Rad27/Fen1, and PCNA in the repair of clustered DNA lesions induced by MMS in G₁ stationary-phase haploid yeast. We found that Pol32 plays an important role in ensuring that clustered lesions are efficiently repaired and do not transition to DSBs.     

How much the eye tells the brain

In the classic "What the frog's eye tells the frog's brain," Lettvin and colleagues showed that different types of retinal ganglion cell send specific kinds of information. For example, one type responds best to a dark, convex form moving centripetally (a fly). Here we consider a complementary question: how much information does the retina send and how is it apportioned among different cell types? Recording from guinea pig retina on a multi-electrode array and presenting various types of motion in natural scenes, we measured information rates for seven types of ganglion cell. Mean rates varied across cell types (6-13 bits . s(-1)) more than across stimuli. Sluggish cells transmitted information at lower rates than brisk cells, but because of trade-offs between noise and temporal correlation, all types had the same coding efficiency. Calculating the proportions of each cell type from receptive field size and coverage factor, we conclude (assuming independence) that the approximately 10(5) ganglion cells transmit on the order of 875,000 bits . s(-1). Because sluggish cells are equally efficient but more numerous, they account for most of the information. With approximately 10(6) ganglion cells, the human retina would transmit data at roughly the rate of an Ethernet connection.