Electron tomography of late stages of FcRn-mediated antibody transcytosis in neonatal rat small intestine

The neonatal Fc receptor (FcRn) transports maternal immunoglobulin (IgG) across epithelia to confer passive immunity to mammalian young. In newborn rodents, FcRn transcytoses IgG from ingested milk across the intestinal epithelium for release into the bloodstream. We used electron tomography to examine FcRn transport of Nanogold-labeled Fc (Au-Fc) in neonatal rat jejunum, focusing on later aspects of transport by chasing Au-Fc before fixation. We observed pools of Au-Fc in dilated regions of the lateral intercellular space (LIS), likely representing exit sites where Au-Fc accumulates en route to the blood. Before weaning, the jejunum functions primarily in IgG transport and exhibits unusual properties: clathrin-rich regions near/at the basolateral LIS and multivesicular bodies (MVBs) expressing early endosomal markers. To address whether these features are related to IgG transport, we examined LIS and endocytic/transcytotic structures from neonatal and weaned animals. Weaned samples showed less LIS-associated clathrin. MVBs labeled with late endosomal/lysosomal markers were smaller than their neonatal counterparts but contained 10 times more internal compartments. These results are consistent with hypotheses that clathrin-rich basolateral regions in neonatal jejunum are involved in IgG exocytosis and that MVBs function in IgG transport while FcRn is expressed but switch to degradative functions after weaning, when the jejunum does not express FcRn or transport IgG

Organization of the Smallest Eukaryotic Spindle

In metazoans, plants, and fungi, the spindle checkpoint delays mitosis until each chromosome is attached to one or more of its own kinetochore microtubules (kMTs). Some unicellular eukaryotes, however, have been reported to have fewer kMTs than chromosomes. If this is the case, it is unclear how the spindle checkpoint could be satisfied. In the vast majority of the previous studies, mitotic cells were chemically fixed at room temperature, but this does not always preserve dynamic and/or small structures like spindle MTs and kinetochores. Indeed, later higher-resolution studies have reversed some earlier claims. Here we show that in Ostreococcus tauri (the smallest eukaryote known), mitosis does involve fewer spindle microtubules than chromosomes. O. tauri cultures were enriched for mitotic cells, high-pressure frozen, and then imaged in 3D both in plastic and in a near-native ("frozen-hydrated") state through electron tomography. Mitotic cells have a distinctive intranuclear heterochromatin-free "spindle tunnel" with approximately four short and occasionally one long, incomplete (unclosed) microtubule at each end of the spindle tunnel. Because other aspects of O. tauri’s spindle checkpoint seem typical, these data suggest that O. tauri’s 20 chromosomes are physically linked and segregated as just one or a small number of groups

Multiscale imaging of HIV-1 transmission in humanized mice

HIV transmission within lymphoid tissues remains incompletely characterized at the level of individual cells and virions. Here we visually describe our approach to understanding HIV-1 dissemination at different levels of volume and resolution within lymphoid tissues from HIV-1-infected humanized mice. We combined tissue clearing techniques, immunostaining, and light sheet fluorescence microscopy to visualize large-volumes of intact tissue with single-cell resolution from HIV-1-infected humanized mice. In parallel, we imaged adjacent regions of tissue using electron microscopy and electron tomography to gain 3D ultrastructural information about the same tissue samples. This approach can provide spatial information about the density and distribution of target cells, HIV-1-infected cells, and individual budding and free-virions within lymphoid tissues. Multiscale imaging of HIV-1 infected tissues from humanized mice can provide insight into the biological mechanisms of HIV-1 transmission through the correlation of global pathology with structural details and these methods are directly translatable to other animal models and human clinical samples

Multiscale Imaging of HIV-1 Transmission in Humanized Mice

HIV transmission within lymphoid tissues remains incompletely characterized at the level of individual cells and virions. Here we visually describe our approach to understanding HIV-1 dissemination at different levels of volume and resolution within lymphoid tissues from HIV-1-infected humanized mice. We combined tissue clearing techniques, immunostaining, and light sheet fluorescence microscopy to visualize large-volumes of intact tissue with single-cell resolution from HIV-1-infected humanized mice. In parallel, we imaged adjacent regions of tissue using electron microscopy and electron tomography to gain 3D ultrastructural information about the same tissue samples. This approach can provide spatial information about the density and distribution of target cells, HIV-1-infected cells, and individual budding and free-virions within lymphoid tissues. Multiscale imaging of HIV-1 infected tissues from humanized mice can provide insight into the biological mechanisms of HIV-1 transmission through the correlation of global pathology with structural details and these methods are directly translatable to other animal models and human clinical samples

The Singlet Contribution to the Structure Function g1(x,Q2)g_1(x,Q^2) at Small xx

The resummation of $O(\alpha_s^{l+1} \ln^{2l} x)$ terms in the evolution equation of the singlet part of $g_1(x,Q^2)$ is carried out. The corresponding singlet evolution kernels are calculated explicitely. The leading small-$x$ contribution to the three-loop splitting function matrix is determined in the $\overline{\rm MS}$ scheme. Relations are derived for the case of ${\cal N} = 1$ supersymmetric Yang--Mills field theory. Numerical results are presented for the polarized singlet and gluon densities, and the structure functions $g_1^{\, p}(x,Q^2)$ and $g_1^{\, n}(x,Q^2)$. They are compared for different assumptions on the non--perturbative input distributions, and the stability of the results against presently unknown subleading contributions is investigated.Comment: 10 pages Latex, including two eps-figures, all compressed by uufil

Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane

The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun's lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer

Architecture and host interface of environmental chlamydiae revealed by electron cryotomography

Chlamydiae comprise important pathogenic and symbiotic bacteria that alternate between morphologically and physiologically different life stages during their developmental cycle. Using electron cryotomography, we characterize the ultrastructure of the developmental stages of three environmental chlamydiae: Parachlamydia acanthamoebae, Protochlamydia amoebophila and Simkania negevensis. We show that chemical fixation and dehydration alter the cell shape of Parachlamydia and that the crescent body is not a developmental stage, but an artefact of conventional electron microscopy. We further reveal type III secretion systems of environmental chlamydiae at macromolecular resolution and find support for a chlamydial needle-tip protein. Imaging bacteria inside their host cells by cryotomography for the first time, we observe marked differences in inclusion morphology and development as well as host organelle recruitment between the three chlamydial organisms, with Simkania inclusions being tightly enveloped by the host endoplasmic reticulum. The study demonstrates the power of electron cryotomography to reveal structural details of bacteria–host interactions that are not accessible using traditional methods