Design principles for selective polarization of PAR proteins by cortical flows.

Clustering of membrane-associated molecules is thought to promote interactions with the actomyosin cortex, enabling size-dependent transport by actin flows. Consistent with this model, in the Caenorhabditis elegans zygote, efficient anterior segregation of the polarity protein PAR-3 requires oligomerization. However, through direct assessment of local coupling between motion of PAR proteins and the underlying cortex, we find no links between PAR-3 oligomer size and the degree of coupling. Indeed, both anterior and posterior PAR proteins experience similar advection velocities, at least over short distances. Consequently, differential cortex engagement cannot account for selectivity of PAR protein segregation by cortical flows. Combining experiment and theory, we demonstrate that a key determinant of differential segregation of PAR proteins by cortical flow is the stability of membrane association, which is enhanced by clustering and enables transport across cellular length scales. Thus, modulation of membrane binding dynamics allows cells to achieve selective transport by cortical flows despite widespread coupling between membrane-associated molecules and the cell cortex.

Patterning and polarization of cells by intracellular flows.

Beginning with Turing's seminal work [1], decades of research have demonstrated the fundamental ability of biochemical networks to generate and sustain the formation of patterns. However, it is increasingly appreciated that biochemical networks also both shape and are shaped by physical and mechanical processes [2, 3, 4]. One such process is fluid flow. In many respects, the cytoplasm, membrane and actin cortex all function as fluids, and as they flow, they drive bulk transport of molecules throughout the cell. By coupling biochemical activity to long-range molecular transport, flows can shape the distributions of molecules in space. Here, we review the various types of flows that exist in cells, with the aim of highlighting recent advances in our understanding of how flows are generated and how they contribute to intracellular patterning processes, such as the establishment of cell polarity.

A cell size threshold limits cell polarity and asymmetric division potential.

Reaction-diffusion networks underlie pattern formation in a range of biological contexts, from morphogenesis of organisms to the polarisation of individual cells. One requirement for such molecular networks is that output patterns be scaled to system size. At the same time, kinetic properties of constituent molecules constrain the ability of networks to adapt to size changes. Here we explore these constraints and the consequences thereof within the conserved PAR cell polarity network. Using the stem cell-like germ lineage of the C. elegans embryo as a model, we find that the behaviour of PAR proteins fails to scale with cell size. Theoretical analysis demonstrates that this lack of scaling results in a size threshold below which polarity is destabilized, yielding an unpolarized system. In empirically-constrained models, this threshold occurs near the size at which germ lineage cells normally switch between asymmetric and symmetric modes of division. Consistent with cell size limiting polarity and division asymmetry, genetic or physical reduction in germ lineage cell size is sufficient to trigger loss of polarity in normally polarizing cells at predicted size thresholds. Physical limits of polarity networks may be one mechanism by which cells read out geometrical features to inform cell fate decisions.

Regulated Activation of the PAR Polarity Network Ensures a Timely and Specific Response to Spatial Cues.

How do cells polarize at the correct time and in response to the correct cues? In the C. elegans zygote, the timing and geometry of polarization rely on a single dominant cue-the sperm centrosome-that matures at the end of meiosis and specifies the nascent posterior. Polarization requires that the conserved PAR proteins, which specify polarity in the zygote, be poised to respond to the centrosome. Yet, how and when PAR proteins achieve this unpolarized, but responsive, state is unknown. We show that oocyte maturation initiates a fertilization-independent PAR activation program. PAR proteins are initially not competent to polarize but gradually acquire this ability following oocyte maturation. Surprisingly, this program allows symmetry breaking even in unfertilized oocytes lacking centrosomes. Thus, if PAR proteins can respond to multiple polarizing cues, how is specificity for the centrosome achieved? Specificity is enforced by Polo-like and Aurora kinases (PLK-1 and AIR-1 in C. elegans), which impose a delay in the activation of the PAR network so that it coincides with maturation of the centrosome cue. This delay suppresses polarization by non-centrosomal cues, which can otherwise trigger premature polarization and multiple or reversed polarity domains. Taken together, these findings identify a regulatory program that enforces proper polarization by synchronizing PAR network activation with cell cycle progression, thereby ensuring that PAR proteins respond specifically to the correct cue. Temporal control of polarity network activity is likely to be a common strategy to ensure robust, dynamic, and specific polarization in response to developmentally deployed cues.

Anterior-enriched filopodia create the appearance of asymmetric membrane microdomains in polarizing C. elegans zygotes.

The association of molecules within membrane microdomains is critical for the intracellular organization of cells. During polarization of the C. elegans zygote, both polarity proteins and actomyosin regulators associate within dynamic membrane-associated foci. Recently, a novel class of asymmetric membrane-associated structures was described that appeared to be enriched in phosphatidylinositol 4,5-bisphosphate (PIP2), suggesting that PIP2 domains could constitute signaling hubs to promote cell polarization and actin nucleation. Here, we probe the nature of these domains using a variety of membrane- and actin cortex-associated probes. These data demonstrate that these domains are filopodia, which are stimulated transiently during polarity establishment and accumulate in the zygote anterior. The resulting membrane protrusions create local membrane topology that quantitatively accounts for observed local increases in the fluorescence signal of membrane-associated molecules, suggesting molecules are not selectively enriched in these domains relative to bulk membrane and that the PIP2 pool as revealed by PHPLCδ1 simply reflects plasma membrane localization. Given the ubiquity of 3D membrane structures in cells, including filopodia, microvilli and membrane folds, similar caveats are likely to apply to analysis of membrane-associated molecules in a broad range of systems.

Asymmetric division of contractile domains couples cell positioning and fate specification.

During pre-implantation development, the mammalian embryo self-organizes into the blastocyst, which consists of an epithelial layer encapsulating the inner-cell mass (ICM) giving rise to all embryonic tissues. In mice, oriented cell division, apicobasal polarity and actomyosin contractility are thought to contribute to the formation of the ICM. However, how these processes work together remains unclear. Here we show that asymmetric segregation of the apical domain generates blastomeres with different contractilities, which triggers their sorting into inner and outer positions. Three-dimensional physical modelling of embryo morphogenesis reveals that cells internalize only when differences in surface contractility exceed a predictable threshold. We validate this prediction using biophysical measurements, and successfully redirect cell sorting within the developing blastocyst using maternal myosin (Myh9)-knockout chimaeric embryos. Finally, we find that loss of contractility causes blastomeres to show ICM-like markers, regardless of their position. In particular, contractility controls Yap subcellular localization, raising the possibility that mechanosensing occurs during blastocyst lineage specification. We conclude that contractility couples the positioning and fate specification of blastomeres. We propose that this ensures the robust self-organization of blastomeres into the blastocyst, which confers remarkable regulative capacities to mammalian embryos.

Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells.

The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015) [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP) in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

The activity and localization patterns of cathepsins B and X in cells of the mouse gastrointestinal tract differ along its length.

Cysteine cathepsins are expressed in most tissues, including the gastrointestinal tract. We demonstrated an involvement of mouse intestinal cathepsin B in extracellular matrix remodeling for regeneration from trauma. The present study aimed at elucidating roles of cysteine cathepsins in the non-traumatized gastrointestinal tract of mice. Thus we investigated expression and localization patterns of cathepsin B and its closest relative, cathepsin X, along the length of the gastrointestinal tract, and determined the effects of their absence. Cathepsin B showed the highest protein levels in the anterior segments of the gastrointestinal tract, whereas the highest activity was observed in the jejunum, as revealed by cathepsin B-specific activity-based probe labeling. Cathepsin X was most abundant in the jejunum and protein levels were elevated in duodenum and colon of Ctsb-/- mice. The segmental pattern of cathepsin expression was reflected by a compartmentalized distribution of junction proteins and basal lamina constituents, changes in tissue architecture and altered activities of the brush border enzyme aminopeptidase N. In conclusion, we observed different compensatory effects and activity levels of cysteine peptidases along the length of the small and large intestines in a segment-specific manner suggesting specific in situ functions of these enzymes in particular parts of the gastrointestinal tract.