Hepatocyte apical bulkheads provide a mechanical means to oppose bile pressure

Abstract

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.

Shaping epithelial lumina under pressure

“…This symmetry breaking could indeed result from pre-existing mechanical asymmetries of the biological system, or asymmetries caused by biological signalling during lumenogenesis. In the early, spherical stage of a forming bile canaliculus [ 35 ], the junction belt constitutes an example of such a mechanical asymmetry [ 21 ], but it remains unknown whether and how this asymmetry is coupled to the sphere-to-tube transition of the bile canaliculus. Moreover, as noted above, during the later extension of tubular bile canaliculi, anisotropic mechanics help to guide the bile canaliculi via anisotropic tension [ 18 ] and support them via apical bulkheads [ 21 , 29 ].…”

“…Hepatocytes from mice and rats also form pressurised lumina in culture [ 21 , 32 , 33 ]. In vivo , these structures extend and connect to form the bile canaliculi network [ 34 , 35 ], which transports bile from the hepatocytes that produce it to the bile ducts which eventually drain the bile into the intestine.…”

“…Structures called ‘apical bulkheads’ can be found in bile canaliculi after the transition into a tube [ 29 ]. While these bulkheads are known to be mechanical elements that increase the pressure that a bile canaliculus can hold [ 21 ], the biological and physical mechanisms of their formation remain a mystery. Understanding more about how bile canaliculi transition to a tubular shape and how apical bulkheads form, therefore, promises key insights into the formation of bile canaliculi in development and into the response of bile canaliculi to bile pressure changes in normal physiology and in disease [ 38 ].…”

Hepatoblast iterative apicobasal polarization is regulated by extracellular matrix remodeling

“…Later, the lumina open and expand due to secretion of osmotically active molecules by the hepatoblasts (Dasgupta, 2018 15 ). The peculiarity of bile canalicular expansion is its extreme anisotropy, which is enforced by mechanical elements named apical bulkheads (Belicova, 2021 16 ; Bebelman 2023 17 ). This expansion results in the formation of narrow tubes.…”

Hepatocyte differentiation requires anisotropic expansion of bile canaliculi

“…We recently described that to form BC, hepatocytes generate transversal apical connections, termed apical bulkheads, that mechanically connect the apical membranes of juxtaposed hepatocytes and enforce the anisotropic expansion of nascent apical lumina into tubular BC (Bebelman et al, 2023, Belicova et al, 2021). Loss of apical bulkheads, through genetic manipulation (see below), results in the isotropic expansion of newly formed apical lumina leading to the formation of multicellular cysts in which the cells adopt a vectorial polarization, similar to cholangiocytes.…”