Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.

Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT ‘trains’, linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of ‘long’ and ‘short’ trains. Here, we analyse the relative number of these trains throughout flagellar regeneration and show that long trains are most abundant at the beginning of flagellar growth whereas short trains gradually increase in number as flagella elongate. These observations are incompatible with the previous hypothesis that short trains are derived solely from the reorganization of long trains at the flagellar tip. We demonstrate with electron tomography the existence of two distinct ultrastructural organizations for the short trains, we name these ‘narrow’ and ‘wide’, and provide the first 3D model of the narrow short trains. These trains are characterized by tri-lobed units, which repeat longitudinally every 16 nm and contact protofilament 7 of the B-tubule. Functional implications of the new structural evidence are discussed.

Embryo implantation has been defined as the “black box” of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.

To analyse whether an HIV-1 envelope protein might play a role in damaging the blood-brain barrier as a fundamental step in the early invasion of the central nervous system by HIV-1. Analysis of permeability of rat brain endothelium cultures to albumin, to assess the functional integrity of the vascular component of the blood-brain barrier. Rat brain endothelium cultures prepared by cerebral microvessels were exposed to recombinant gp120IIIB on microporous membranes and passage of biotin-labelled albumin was analysed. Scanning electron microscopy was used to analyse cell culture morphology. Some cultures were preincubated with N-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of nitric oxide synthase, or with spantide, a selective substance P antagonist. HIV-1 gp120 increased the permeability of rat brain endothelial cells to albumin in a dose-dependent manner. Scanning electron microscopy revealed profound gp120-induced alterations in cell morphology accounting for the increased permeability to macromolecules. These alterations were neutralized by anti-gp120 monoclonal antibody but not by isotype control antibody or L-NAME. By contrast, spantide and anti-substance P polyclonal antibody completely blocked the gp120-induced increase in albumin permeability. Control cultures exposed to measles virus nucleoprotein showed an increase in permeability that was not blocked by spantide. Brain endothelial cells, exposed to gp120, displayed cell surface immunoreactivity for substance P, suggesting that substance P is secreted by brain endothelium in response to gp120 stimulation and binds to brain endothelial cells through a receptor-mediated mechanism. These findings suggest a role for substance P in the gp120-induced increase in permeability of brain endothelium.