Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position

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

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

The serine/threonine kinase Par1b regulates epithelial lumen polarity via IRSp53-mediated cell–ECM signaling

Par1b regulates cell–ECM signaling and dictates epithelial lumenal organization by targeting IRSp53, a Rho GTPase adaptor and scaffolding protein

Steady state concentrations of three classes of enzymes in the unrestricted fusion scenario.

<p>Golgi enzyme concentrations are normalized by their maximum value. The parameters are: Decay rates for the <i>cis</i> and <i>trans</i> t-SNARE are , the vesicular transport coefficient , and dissociation constants for vesicular binding are: for the ER v-SNARE, for the <i>cis</i> t- and v-SNAREs, for <i>trans</i> t-SNARE, for <i>trans</i> v-SNARE, for <i>cis</i> enzymes, for medial enzymes, and for <i>trans</i> enzymes. Initial concentrations of all proteins in the first cisterna are , and the concentration of the t-SNARE in the ER is 0.7.</p

Schematic representation of a stacked Golgi apparatus that undergoes cisternal maturation.

<p>A) ER-derived vesicles (beige) fuse with each other to yield the first, most <i>cis</i>, cisterna. Individual cisterna mature from position 1 to position 8, where they disintegrate into transport carriers destined for the plasma membrane and endosomes. Vesicles originating from cisterna #2 deliver <i>cis</i> Golgi proteins to cisterna #1 while at the same time cisterna #2 receives Golgi resident proteins from cisterna #3. B) The cisternae are categorized as <i>cis</i>, medial and <i>trans</i> based on the abundance of Golgi residence proteins, mostly glycosylating enzymes, which exhibit distinct but overlapping peaks along the Golgi stack according to their sequential role in the processing of exocytic cargo. C) Two SNARE pairs, which we term SNARE (purple) and SNARE (green) are thought to mediate intra-Golgi transport of resident proteins. The respective v and t-SNAREs of SNARE both decay with a steep gradient from <i>cis</i> to <i>trans</i>. -t-SNAREs decay with a shallow gradient, while its corresponding -v-SNARE concentration increases from cisternae 1 to 8. The graphs are schematic representations of data from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003125#pcbi.1003125-Volchuk1" target="_blank">[17]</a>.</p

Distribution of SNARE for large number of cisternae.

<p>The steady state gradient has the exponentially decaying form, where depends on two dimensionless groups of parameters, which caracterizes the decay of the SNARE and which characterizes the vesicular transport of SNARE: and (solid line) with the best fit given by , and (dashed line) with the best fit given by . Theoretical values of determined from (11) are indistinguishable from the values obtained as the best fit for the simulations. The dotted line corresponds to the case of zero loss, and , and illustrates the absence of a concentration gradient. To reveal the exponential decay of the SNARE concentration, we purposefully consider a non-biologically high number of compartments and analyze the SNARE concentration away from both the <i>cis</i> and <i>trans</i> ends of the stack, where the boundary effects can play a role.</p

Self-generated concentrations of SNAREs and enzymes.

<p><b>Panel A:</b> Steady state concentration of cisternal SNARE vs the number of cisterna for: both the loss and the vesicular transport mechanisms are enacted (solid line), only the loss mechanism operates (dashed line), only vesicular transport occurs (dotted line). All concentrations are sampled immediately before the cisternal shift event, when the number of each cisterna is incremented by one. The definitions of parameters are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003125#s4" target="_blank">Methods</a>. Here and in all following plots it is assumed that and , i.e. all concentrations are expressed in the units of initial concentrations and the time is expressed in units of the cisternal maturation period. Solid line: and , dashed line: and , dotted line: and , for all curves . <b>Panel B:</b> Distribution of Golgi enzymes: <i>cis</i> (solid line), medial (dashed line) and <i>trans</i> (dotted line) established as a result of competition for incorporation into vesicles. Vesicular flux is controlled by the gradient of cisternal SNAREs shown by the solid line in the left panel, vesicles from the first cisterna can exit the Golgi and fuse with the ER. The parameters for the enzyme transport are , , , and .</p

Dissociation constants for binding to vesicular sites that yield the plots depicted in Fig. 3.

<p>Dissociation constants for binding to vesicular sites that yield the plots depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003125#pcbi-1003125-g003" target="_blank">Fig. 3</a>.</p