Nano-scale architecture of blood-brain barrier tight-junctions

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: No explicit power analysis was used; sample size for all immunofluorescence experiments was determined empirically using standards generally employed by the field: a minimum of three animals per group in each experiment, a minimum of four tissue sections of each tissue and a minimum of 10 capillaries per group. Information of exact repetitions and sample numbers can be found in figure legends. Information of exact repetitions and sample numbers can be found in figure legends. In general: For in vitro studies: 5 independent experiments were preformed in which every experiment was a different sub-culture passage preformed in a separate day (considered as a biological replicate). For technical replications (Figure 1), a minimum of 4 microscopic fields produced n=183 clusters (post-confluence) and 281 clusters (super-confluence). We excluded no outliers. For in vivo studies: data from minimum three animals per group in each experiment were considered biological replicates. For technical replications a set of minimum 3 tissue sections of each animal produced: 1. Clustering properties (Figure 2, S1) -a set of 20 capillaries of each age (E12, P9), 657 clusters (E12)

Statistical reporting • Statistical analysis methods should be described and justified
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Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Information of exact repetitions and sample numbers (raw data) is presented in figure legends, including statistical analysis methods of each experiment, definition of center (mean±s.e.m). These appear also in the Statistical analysis section in the methods section -All comparisons were performed by two-tailed Mann-Whitney U-tests, P<0.05 was considered significant (GraphPad Prism 8.0.1(244) for Windows, GraphPad Software, San diego, California, USA). Reports of exact p-values wherever possible appear in the text.
In tissues form wild-type mice samples were allocated based on the embryonic age (indicated in the text/methods and figure legends). In images from in vitro experiments samples were allocated based on the culture age (indicated in the text/methods and figure legends). In the data set of claudin-5 null and control littermates, the person collecting the data and analyzing was blind to the animal's genotype (in the Statistical analysis section in the methods section).
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