Characterisation of Novel Rab5 Effector Proteins in the Endocytic Pathway

Endocytosis, a process of plasma membrane invaginations, is a fundamental cellular mechanism, ensuring uptake of nutrients, enhanced communication between cells, protective functions against invasive pathogens and remodelling of the plasma membrane composition. In turn, endocytic mechanisms are exploited by pathogens to enter their host cells. Endocytosis comprises multiple forms of which our molecular understanding has mostly advanced with respect to clathrin-mediated endocytosis and phagocytosis. Studies on the small GTPase Rab5 have provided important insights into the molecular mechanism of endocytosis and transport in the early stages of the endocytic pathways. Rab5 is a key regulator of clathrin-mediated endocytosis, but in addition, localises to several distinct endocytic carriers including phagosomes and pinocytic vesicles. On early endosomes, Rab5 coordinates within a spatially restricted domain enriched in phosphatidylinositol-3 phosphate PI(3)P a complex network of effectors, including PI3-Kinase (PI3-K), the FYVE-finger proteins EEA1 and Rabenosyn-5 that functionally cooperate in membrane transport. Moreover, Rab5 regulates endocytosis from the apical and basolateral plasma membrane in polarised epithelial cells. During my PhD thesis, I investigated the molecular mechanisms of endocytosis both in polarised and non-polarised cells. I obtained new insights into the molecular mechanisms of endocytosis and their coordination through the functional characterization of a novel Rab5 effector, termed Rabankyrin-5. I could demonstrated that Rabankyrin-5 is a novel PI(3)P-binding Rab5 effector that localises to early endosomes and stimulates their fusion activity in vitro. The latter activity depends on the oligomerisation of Rabankyrin-5 on the endosomal membrane via the N-terminal BTB/POZ domain. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid phase uptake whereas its downregulation through RNA interference inhibits these processes. In polarised epithelial cells, the function of Rabankyrin-5 is primarily restricted to the apical membrane. It localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells and its overexpression in polarised MDCK cells specifically stimulates apical but not basolateral, non-clathrin mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)-pinocytosis, my studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, the active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells. The results obtained in this thesis are central not only for our understanding of the basic principles underlying the regulation of multiple endocytic mechanisms. They are also relevant for the biomedical field, since actin-dependent (macro)-pinocytosis is an important mechanism for the physiology of cells and organisms and is upregulated under certain pathological conditions (e.g. cancer)

The Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells

The PCP genes Celsr1 and Vangl2 are required for normal lung branching morphogenesis

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1Crsh and Vangl2Lp mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies

Characterisation of Novel Rab5 Effector Proteins in the Endocytic Pathway

Endocytosis, a process of plasma membrane invaginations, is a fundamental cellular mechanism, ensuring uptake of nutrients, enhanced communication between cells, protective functions against invasive pathogens and remodelling of the plasma membrane composition. In turn, endocytic mechanisms are exploited by pathogens to enter their host cells. Endocytosis comprises multiple forms of which our molecular understanding has mostly advanced with respect to clathrin-mediated endocytosis and phagocytosis. Studies on the small GTPase Rab5 have provided important insights into the molecular mechanism of endocytosis and transport in the early stages of the endocytic pathways. Rab5 is a key regulator of clathrin-mediated endocytosis, but in addition, localises to several distinct endocytic carriers including phagosomes and pinocytic vesicles. On early endosomes, Rab5 coordinates within a spatially restricted domain enriched in phosphatidylinositol-3 phosphate PI(3)P a complex network of effectors, including PI3-Kinase (PI3-K), the FYVE-finger proteins EEA1 and Rabenosyn-5 that functionally cooperate in membrane transport. Moreover, Rab5 regulates endocytosis from the apical and basolateral plasma membrane in polarised epithelial cells. During my PhD thesis, I investigated the molecular mechanisms of endocytosis both in polarised and non-polarised cells. I obtained new insights into the molecular mechanisms of endocytosis and their coordination through the functional characterization of a novel Rab5 effector, termed Rabankyrin-5. I could demonstrated that Rabankyrin-5 is a novel PI(3)P-binding Rab5 effector that localises to early endosomes and stimulates their fusion activity in vitro. The latter activity depends on the oligomerisation of Rabankyrin-5 on the endosomal membrane via the N-terminal BTB/POZ domain. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid phase uptake whereas its downregulation through RNA interference inhibits these processes. In polarised epithelial cells, the function of Rabankyrin-5 is primarily restricted to the apical membrane. It localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells and its overexpression in polarised MDCK cells specifically stimulates apical but not basolateral, non-clathrin mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)-pinocytosis, my studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, the active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells. The results obtained in this thesis are central not only for our understanding of the basic principles underlying the regulation of multiple endocytic mechanisms. They are also relevant for the biomedical field, since actin-dependent (macro)-pinocytosis is an important mechanism for the physiology of cells and organisms and is upregulated under certain pathological conditions (e.g. cancer)

Characterisation of Novel Rab5 Effector Proteins in the Endocytic Pathway

Endocytosis, a process of plasma membrane invaginations, is a fundamental cellular mechanism, ensuring uptake of nutrients, enhanced communication between cells, protective functions against invasive pathogens and remodelling of the plasma membrane composition. In turn, endocytic mechanisms are exploited by pathogens to enter their host cells. Endocytosis comprises multiple forms of which our molecular understanding has mostly advanced with respect to clathrin-mediated endocytosis and phagocytosis. Studies on the small GTPase Rab5 have provided important insights into the molecular mechanism of endocytosis and transport in the early stages of the endocytic pathways. Rab5 is a key regulator of clathrin-mediated endocytosis, but in addition, localises to several distinct endocytic carriers including phagosomes and pinocytic vesicles. On early endosomes, Rab5 coordinates within a spatially restricted domain enriched in phosphatidylinositol-3 phosphate PI(3)P a complex network of effectors, including PI3-Kinase (PI3-K), the FYVE-finger proteins EEA1 and Rabenosyn-5 that functionally cooperate in membrane transport. Moreover, Rab5 regulates endocytosis from the apical and basolateral plasma membrane in polarised epithelial cells. During my PhD thesis, I investigated the molecular mechanisms of endocytosis both in polarised and non-polarised cells. I obtained new insights into the molecular mechanisms of endocytosis and their coordination through the functional characterization of a novel Rab5 effector, termed Rabankyrin-5. I could demonstrated that Rabankyrin-5 is a novel PI(3)P-binding Rab5 effector that localises to early endosomes and stimulates their fusion activity in vitro. The latter activity depends on the oligomerisation of Rabankyrin-5 on the endosomal membrane via the N-terminal BTB/POZ domain. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid phase uptake whereas its downregulation through RNA interference inhibits these processes. In polarised epithelial cells, the function of Rabankyrin-5 is primarily restricted to the apical membrane. It localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells and its overexpression in polarised MDCK cells specifically stimulates apical but not basolateral, non-clathrin mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)-pinocytosis, my studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, the active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells. The results obtained in this thesis are central not only for our understanding of the basic principles underlying the regulation of multiple endocytic mechanisms. They are also relevant for the biomedical field, since actin-dependent (macro)-pinocytosis is an important mechanism for the physiology of cells and organisms and is upregulated under certain pathological conditions (e.g. cancer)

Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

<div><p>The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.</p> </div

Line3-2 mutation perturbs cell proliferation and causes increased apoptosis in the lung.

<p>(A–I) In mutant lungs, cell proliferation is decreased during the pseudoglandular stage in mutant lungs, but is higher at the terminal sac stage as detected by EdU incorporation in lung sections. (I) Data presented as percentage of total nuclei. The results are representatives of 6 different sections. (J–R) Apoptosis is markedly increased in mutant lungs. (J–R) Lungs from E14.5 embryos were sectioned and processed for TUNEL staining. (R) The number of TUNEL positive cells were determined on sections and presented as percentage of total nuclei. The results are representatives of 7 different sections. Error bars are presented as SEM. (High) indicates higher magnification view.</p

Line3-2 contains two missense mutations in the Nubp1 gene.

<p>(A) Schematic representation of the genomic region around the Nubp1 gene, which contains two missense mutations at nucleotide position 743 and 803 of the coding sequence. The mutations leads to a Threonine to Lysine and Proline to Glutamine change in the amino acid sequence of Nubp1. (B) Protein sequence alignment of the indicated genes show the mutations in line3-2 to be in a highly conserved region. Red boxes and asterisks highlight mutated regions. (C–H) E14.5 embryos from a complementation cross between line3-2 and Nubp1 knock-out reveals similar defects in lung (H) and limb development (G) as lin3-2 homozygous mutants (E, F).</p

Nubp1^(m1Nisw) lungs show altered distribution of Par3 and Numb1.

<p>(A–R) E16.5 lungs were sectioned and stained by immunohistochemistry for the indicated proteins. (A–H) The cellular localization of the polarity protein Par3 is significantly disrupted in Nubp1^(m1Nisw) mutant lung. (I–R) The mutation in Nubp1^(m1Nisw) disrupts the cellular localization of Numb1. Note the defect is restricted to distal epithelial cells. Images K-R are higher magnifications of the regions indicated in images I and J.</p

Line3-2 reveals lung hypoplasia and limb syndactyly.

<p>(A–H) Embryos dissected at E18.5 (A) reveal that line3-2 mutants are slightly reduced in size and have significantly smaller lung lobes (B–F). (G–H) E18.5 embryos fixed and stained for bone (red) and cartilage (blue) in the forelimb demonstrate gross malformation and reduction in the number of digits in the line3-2. (I–N) Histological analysis of E18.5 lung lobes show decreased airway number, increased airway diameter and defects in distal lung epithelial morphology in line3-2. (I–L) E18.5 lungs were dissected, paraffin embedded and sectioned at 10 um. (M) Airway number and (N) the mean linear intercept (MLI) of wild-type and mutant lungs were determined from 4 different embryos at E18.5. Error bars are indicated as standard error of the mean (SEM).</p