Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes

Abstract

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct "apicolateral" subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)-positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma.

Figure 1. Schematic overview of the difference between columnar and hepatic epithelium.

(A) Schematic overview of hepatic epithelium and polarity. Note the presence of an apicolateral plasma membrane domain in this type of epithelium. (B) Schematic overview of columnar (i.e., “simple”) epithelium and polarity. (C) Schematic representation of the tissue architecture resulting from symmetric and asymmetric segregation of the apical surface in hepatic epithelium. (D) Schematic representation of the tissue architecture resulting from symmetric and asymmetric segregation of the apical surface in columnar epithelium. The dashed line represents the cleavage furrow and the position of the newly formed membrane. L, lumen; N, nucleus.

Figure 2. Rat and mouse hepatocytes predominantly orient their mitotic spindle axis towards the apicolateral subdomain.

(A) Hepatocytes from mouse livers 48 h post-hepatectomy orient their spindle poles (labeled with NuMA) towards the apicolateral subdomain. (B) Quantification of (A) (n = 61). Dividing hepatocytes predominantly orient their SA towards the apicolateral subdomain. (C) Dividing hepatocytes from weaned rat livers orient their spindle poles (marked by NuMA) towards the apicolateral subdomain (marked by DPPIV and ABCC2) in metaphase and telophase. (D) Apicolateral localization of LGN (white outline arrowheads) in dividing rat liver hepatocytes. The apical domain is labeled with ABBC2. Tight junctions are labeled with ZO-1. The outline diagrams (“PM domains”) show the identity of the cell membranes of the dividing cells (#) shown in (D). Grey, red, orange, and green lines represent the basal, apical, lateral, and apicolateral plasma membrane domains, respectively. All figures: filled white arrowheads mark the bile canaliculus or apical domain. Dotted white lines outline the sinusoid (si). Dashed lines indicate the SA. *p<0.05 (calculated using a paired two-tailed Student's t-test). Scale bars: 5 µm.

Figure 3. Hepatocytes predominantly orient their mitotic spindle axis towards the apicolateral subdomain.

(A) SA axes were quantified as crossing (marked by black arrowheads) the apicolateral membrane (situation 1) or other membranes (situation 2), indicating a bias of the SA axis to cross the apicolateral membrane. (B) Localization of LGN (white outline arrowheads) in polarized HepG2 cells. The apical domain is labeled with ABCB1 and marked by a red arrowhead. The outline diagram (“PM domains”) shows the identity of the cell membranes of the dividing cell (#). Grey, red, orange, and green lines represent the basal, apical, lateral, and apicolateral plasma membrane domains, respectively. (C) Schematic overview of how the orientation of the mitotic spindle (angle between the SA and PA [angle SA/PA]) was measured (see Materials and Methods). (D) Dot plot of the SA/PA angle for dividing HepG2 cells in metaphase. Shown is mean (green bar) and standard error of the mean (SEM) (blue error bars). (E) Histogram analysis reveals a strong bias for HepG2 cells to divide with an SA/PA angle between 0° and 30° during metaphase. *p<0.05; **p<0.01. BC, bile canaliculus. Scale bars: 5 µm.

Figure 4. Hepatocytes segregate the apical plasma membrane and lumen asymmetrically during mitosis.

(A) Stills from Movie S3 showing asymmetric segregation of the apical plasma membrane (ABCB1-eGFP, red arrowheads) in dividing HepG2 cells. Black arrowheads mark the ingressing cleavage furrow during cytokinesis. “1” marks the daughter cell inheriting the apical domain, and “2” marks the daughter cell not inheriting the apical domain, hence becoming non-polarized. (B) Quantification of the asymmetry of apical plasma membrane inheritance in dividing HepG2 cells (live imaging; n = 64). (C) Stills from Movie S5 showing asymmetric segregation of the apical domain (ABCB1-eGFP, red arrowheads) and formation of a new apical domain by the new daughter cell. White arrowheads mark the ingressing cleavage furrow. The yellow arrowhead marks the de novo formed apical domain at the site of cytokinesis. *p<0.05. Scale bars: 5 µm.

Figure 5. Par1b stimulates apicolateral-directed spindle orientation and asymmetric segregation of the apical domain in MDCK cells.

(A) Schematic overview of the polarity phenotype in control and Par1b-overexpressing MDCK cells. (B) Fixed control and MDCK-Par1b cells were labeled for the apical marker gp135 (left panel, red) and LGN (right panel, yellow). Par1b-overexpressing MDCK cells asymmetrically segregate their apical domain (left panel, red arrowheads; black arrowheads mark the ingressing cleavage furrow) during cell division. LGN localizes to the apicolateral plasma membrane domain in Par1b-overexpressing MDCK cells (right panel, black arrowheads). The dashed line marks the common lateral plasma membrane domain. (C) Histogram analysis of the SA/PA angle shows that dividing MDCK-Par1b cells exhibit a bias towards lower angles (0–30°) in metaphase, as observed for hepatocytes. All figures: red arrowheads mark the apical domain. *p<0.05. ***p<0.001. Scale bars: 5 µm.

Figure 6. LGN accumulates at the apicolateral subdomain in a Par1b-dependent manner and controls spindle orientation.

(A and B) Localization of LGN (white outline arrowheads) in control (scrambled) and Par1b shRNA HepG2 cells. The apical domain (red arrowheads) is labeled with ABCC2 (A) or F-actin (B). Tight junctions are labeled with ZO-1 (B). The dashed line represents the SA. The solid line represents the PA. The outline diagrams (“PM domains”) show the identity of the cell membranes of the dividing cells (#). Grey, red, orange, and green lines represent the basal, apical, lateral, and apicolateral plasma membrane domains, respectively. (C) Western blot analysis of LGN knockdown in HepG2 cells using two shRNA constructs. (D) Histogram analysis of SA/PA angles in LGN knockdown HepG2 cells indicating a loss of bias towards lower angles (0–30°) under LGN knockdown conditions. (E) Histogram analysis of wild type, control (scrambled) shRNA, Par1b knockdown, and Par1b rescued HepG2 cells shows a loss of apicolateral-directed spindle orientation under Par1b knockdown conditions. *p<0.05. **p<0.01. n.s., not significant. Scale bars: 5 µm.

Figure 7. Implications of mitotic spindle orientation during the development of the unique liver architecture.

(1) LGN localizes to the apicolateral plasma membrane area during hepatocyte cell division. (2) The mitotic spindle orients one of its (NuMA-containing) spindle poles towards the LGN-enriched apicolateral plasma membrane. (3) This orientation of the mitotic spindle results in the cleavage furrow not bisecting the apical plasma membrane, resulting in asymmetric segregation of the apical plasma membrane. (4) New apical surfaces are created de novo at the site of abscission. (5) During early liver development, apical pockets are created between hepatocytes. (6) These pockets grow out to bile canalicular/channel-like structures during later phases of liver development. (7) When Par1b is impaired, LGN migrates away from the apicolateral plasma membrane area and is subsequently found on basal or lateral membranes. (8) The mitotic spindle orients its poles towards LGN-enriched cortical areas. (9) The cleavage furrow has an increased chance of bisecting the apical plasma membrane, resulting in symmetric segregation of the apical plasma membrane. (10) Both cells now share the same apical surface (“simple” epithelial polarity). (11) Continued cell division likely results in the generation of “simple” epithelial cyst-like structures.