Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4^−/− mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4^−/− mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4^−/− mice were injected subretinally with 1 μl (∼5.0 × 10⁵ TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4^−/− mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4^−/− mice accumulated 8–12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3–5 times more A2E (27–39 pmol per eye, s.d.=1.5; P=0.001–0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. coli than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins. Cell division in Escherichia coli begins with the assembly of FtsZ proteins into a ring-like structure, the Z-ring. Remarkably, the Z-ring localizes with very high precision at midcell. Currently, two molecular systems, nucleoid occlusion and the Min system, are known to localize the Z-ring. Here, we explore whether there are additional divisome localization systems in E. coli. Using quantitative fluorescence imaging, we show that slow growing cells lacking both known positioning systems continue to divide accurately at midcell. We find that the terminus region of the chromosome moves first to mid-cell where it functions as a positional landmark for the subsequent localization of the Z-ring. Furthermore, we provide evidence that this divisome positioning system involves MatP, ZapB, and ZapA proteins. Our work shows that E. coli can divide without the canonical mechanisms for localizing its cytokinetic ring. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins.

The microfluidic mother machine platform has attracted much interest for its potential in studies of bacterial physiology, cellular organization, and cell mechanics. Despite numerous experiments and development of dedicated analysis software, differences in bacterial growth and morphology in narrow mother machine channels compared to typical liquid media conditions have not been systematically characterized. Here we determine changes in E. coli growth rates and cell dimensions in different sized dead-end microfluidic channels using high resolution optical microscopy. We find that E. coli adapt to the confined channel environment by becoming narrower and longer compared to the same strain grown in liquid culture. Cell dimensions decrease as the channel length increases and width decreases. These changes are accompanied by increases in doubling times in agreement with the universal growth law. In channels 100 μm and longer, cell doublings can completely stop as a result of frictional forces that oppose cell elongation. Before complete cessation of elongation, mechanical stresses lead to substantial deformation of cells and changes in their morphology. Our work shows that mechanical forces rather than nutrient limitation are the main growth limiting factor for bacterial growth in long and narrow channels.

To successfully propagate, cells need to coordinate chromosomal replication and segregation with cell division to prevent formation of DNA-less cells and cells with damaged DNA. Here, we review molecular systems in Escherichia coli that are known to be involved in positioning the divisome and chromosome relative to each other. Interestingly, this well-studied micro-organism has several partially redundant mechanisms to achieve this task; none of which are essential. Some of these systems determine the localization of the divisome relative to chromosomes such as SlmA-dependent nucleoid occlusion, some localize the chromosome relative to the divisome such as DNA translocation by FtsK, and some are likely to act on both systems such as the Min system and newly described Ter linkage. Moreover, there is evidence that E. coli harbors other divisome-chromosome coordination systems in addition to those known. The review also discusses the minimal requirements of coordination between chromosomes and cell division proteins needed for cell viability. Arguments are presented that cells can propagate without any dedicated coordination between their chromosomes and cell division machinery at the expense of lowered fitness.