Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation.

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

Retrofusion of intralumenal MVB membranes parallels viral infection and coexists with exosome release.

The endosomal system constitutes a highly dynamic vesicle network used to relay materials and signals between the cell and its environment.1 Once internalized, endosomes gradually mature into late acidic compartments and acquire a multivesicular body (MVB) organization through invagination of the limiting membrane (LM) to form intraluminal vesicles (ILVs).2 Cargoes sequestered into ILVs can either be delivered to lysosomes for degradation or secreted following fusion of the MVB with the plasma membrane.3 It has been speculated that commitment to ILVs is not a terminal event, and that a return pathway exists, allowing "back-fusion" or "retrofusion" of intraluminal membranes to the LM.4 The existence of retrofusion as a way to support membrane equilibrium within the MVB has been widely speculated in various cell biological contexts, including exosome uptake5 and major histocompatibility complex class II (MHC class II) antigen presentation.6-9 Given the small physical scale, retrofusion of ILVs cannot be measured with conventional techniques. To circumvent this, we designed a chemically tunable cell-based system to monitor retrofusion in real time. Using this system, we demonstrate that retrofusion occurs as part of the natural MVB lifestyle, with attributes parallel to those of viral infection. Furthermore, we find that retrofusion and exocytosis coexist in an equilibrium, implying that ILVs inert to retrofusion comprise a significant fraction of exosomes destined for secretion. MVBs thus contain three types of ILVs: those committed to lysosomal degradation, those retrofusing ILVs, and those subject to secretion in the form of exosomes. VIDEO ABSTRACT.

Discovering fiber type architecture over the entire muscle using data-driven analysis.

Skeletal muscle function is inferred from the spatial arrangement of muscle fiber architecture, which corresponds to myofiber molecular and metabolic features. Myofiber features are often determined using immunofluorescence on a local sampling, typically obtained from a median region. This median region is assumed to represent the entire muscle. However, it remains largely unknown to what extent this local sampling represents the entire muscle. We present a pipeline to study the architecture of muscle fiber features over the entire muscle, including sectioning, staining, imaging to image quantification and data-driven analysis with Myofiber type were identified by the expression of myosin heavy chain (MyHC) isoforms, representing contraction properties. We reconstructed muscle architecture from consecutive cross-sections stained for laminin and MyHC isoforms. Examining the entire muscle using consecutive cross-sections is extremely laborious, we provide consideration to reduce the dataset without loosing spatial information. Data-driven analysis with over 150,000 myofibers showed spatial variations in myofiber geometric features, myofiber type, and the distribution of neuromuscular junctions over the entire muscle. We present a workflow to study histological changes over the entire muscle using high-throughput imaging, image quantification, and data-driven analysis. Our results suggest that asymmetric spatial distribution of these features over the entire muscle could impact muscle function. Therefore, instead of a single sampling from a median region, representative regions covering the entire muscle should be investigated in future studies.

Mobile late endosomes modulate peripheral endoplasmic reticulum network architecture.

The endoplasmic reticulum (ER) is the largest organelle contacting virtually every other organelle for information exchange and control of processes such as transport, fusion, and fission. Here, we studied the role of the other organelles on ER network architecture in the cell periphery. We show that the co-migration of the ER with other organelles, called ER hitchhiking facilitated by late endosomes and lysosomes is a major mechanism controlling ER network architecture. When hitchhiking occurs, emerging ER structures may fuse with the existing ER tubules to alter the local ER architecture. This couples late endosomal/lysosomal positioning and mobility to ER network architecture. Conditions restricting late endosomal movement-including cell starvation-or the depletion of tether proteins that link the ER to late endosomes reduce ER dynamics and limit the complexity of the peripheral ER network architecture. This indicates that among many factors, the ER is controlled by late endosomal movement resulting in an alteration of the ER network architecture.