PAR-3 mediates the initial clustering and apical localization of junction and polarity proteins during C. elegans intestinal epithelial cell polarization

Achilleos, Annita; Wehman, Ann M.; Nance, Jeremy

doi:10.1242/jcs.074609

Cited by 37 publications

(97 citation statements)

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“…In addition to interacting with PTEN, Baz/PAR-3 also interacts with components of the nectin-and cadherin-based adhesion machinery to promote cadherin clustering, a well-recognized determinant of cadherin adhesive strengthening (Leckband and Prakasam 2006;Harris and Tepass 2010). During the early stages of intestinal epithelial cell polarization in C. elegans, cortical foci containing PAR-3 recruit E-cadherin, PAR-6, and aPKC (Achilleos et al 2010). Furthermore, appropriate positioning of Baz is critical to defining the apical/lateral border (Morais-deSa et al 2010).…”

Section: Pi Control Of Junctional Stabilitymentioning

confidence: 99%

Phosphoinositides in Cell Architecture

Shewan

Eastburn²,

Mostov

2011

Cold Spring Harbor Perspectives in Biology

173

159

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Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.P hosphoinositides (PIs) are essential components of cellular membranes in eukaryotes. Though these specialized lipids comprise less than 1% of the cellular lipid cohort, they play key roles in many fundamental biological processes (Di Paolo and De Camilli 2006;Saarikangas et al. 2010). PIs possess such far ranging roles by serving as specialized membrane docking sites for effectors of numerous cellular signal transduction cascades. PIs also serve as precursors of lipid second messengers. They are concentrated on the cytosolic face of cellular membranes (Fig. 1A) and rapidly diffuse within the plane of the membrane. Reversible phosphorylation of the myo-inositol head group of phosphatidylinositol (PtdIns) at positions 3, 4, and 5 (Fig. 1B) gives rise to the seven PI isoforms identified in eukaryotic cells. PtdIns(4)P and PtdIns(4,5)P 2 are constitutively present in membranes and comprise the largest pool of cellular PIs, whereas PtdIns(3,4,5)P 3 is essentially undetectable in most types of unstimulated cells (Lemmon and Ferguson 2000;Saarikangas et al. 2010).The spatiotemporally regulated production and turnover of phosphoinositides is crucial for localized PI signaling and function. Numerous phosphatidylinositol kinases and phosphatases are involved in regulating the metabolism of the various PI isoforms (Fig. 1)

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Section: Pi Control Of Junctional Stabilitymentioning

confidence: 99%

Phosphoinositides in Cell Architecture

Shewan

Eastburn²,

Mostov

2011

Cold Spring Harbor Perspectives in Biology

173

159

View full text Add to dashboard Cite

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“…This protocol also takes~2 weeks to complete which makes the preparation of transgenes fairly rapid. The protocol has also been tried by other labs with success 20 . 2.…”

Section: Representative Resultsmentioning

confidence: 99%

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

Zhang

Kashyap

Ferguson

et al. 2011

JoVE

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The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method. Protocol OverviewMany transgenes used in the generation of transgenic C. elegans consist of promoter sequences and perhaps a gene cDNA cloned into one of the vectors generated by the lab of Dr. Andy Fire 1 . While these transgenes are often successful with regards to producing a GFP reporter gene or expressing a cDNA in a desired pattern, these transgenes can lack the alternate promoters, enhancer elements, and 3' untranslated region (UTR) elements which play important roles in the control of gene expression in vivo 2 . For example, both the daf-12 and fah-1 genes have important enhancer elements which lie outside of the proximal promoter which were missed in promoter only constructs 3,4,5 . Further many transgene constructs use the unc-54 3'UTR which prevents regulation by the appropriate microRNA genes 6,7,8 . Consequently, generating transgenes with large segments of worm genomic DNA would be ideal for capturing all of promoters, splice variants, and 3' UTR control elements. Recently a C. elegans fosmid library which consists of~40 kb regions of genomic ...

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“…In brief, these events include the correct specification and asymmetric division of the intestinal founder cell EMS (Bossinger and Schierenberg, 1996;Goldstein, 1992;Han, 1997;Kormish et al, 2010;Schierenberg, 1987;see Fig.2 for further details), the ingression of the intestinal precusor cell Ea and Ep during gastrulation ( Fig.5B-C ;Chisholm, 2006;Putzke and Rothman, 2003;Rohrschneider and Nance, 2009;Sawyer et al, 2009;Schierenberg, 2005;Schierenberg, 2006), the cytoplasmic polarization of intestinal primordial cells ( Fig.5D; Achilleos et al, 2010;Bossinger et al, 2001;Leung et al, 1999;Totong et al, 2007), the formation of apical adherens junction and the generation of the future lumen within the primordium (Fig.5E'-E''; Leung et al, 1999), the intercalation of specific sets of cells (Hoffmann et al, 2010;Leung et al, 1999), the invariant 'twist' in the anterior of the intestinal primordium (Hermann et al, 2000), and finally the differentiation of the late embryonic, larval and adult intestine that has been proposed to be under the control of the GATA-factor ELT-2 (McGhee et al, 2009;McGhee et al, 2007;Pauli et al, 2006).…”

Section: Development and Differentiation Of The C Elegans Embryonic mentioning

confidence: 99%

“…The CeAJ is a belt-like junctional structure that encircles the apex of polarized epithelial cells and resembles the ZA in other systems. By immunohistochemistry, the apicolateral membrane domain can be subdivided into four subdomains (Fig.6): the PPC together with the Drosophila Crumbs homolog CRB-1 and the multi PDZ-domain containing protein MAGI-1 (Achilleos et al, 2010;Aono et al, 2004;Bossinger et al, 2001;Stetak and Hajnal, 2011;Totong et al, 2007), the catenin-cadherin complex (CCC; Costa et al, 1998;Grana et al, 2010;Kwiatkowski et al, 2010), the DLG-1-AJM-1 complex (DAC; Bossinger et al, 2001;Firestein and Rongo, 2001;Köppen et al, 2001;Lockwood et al, 2008;McMahon et al, 2001) and the LET-413 protein Legouis et al, 2000;Legouis et al, 2003;Lockwood et al, 2008;Pilipiuk et al, 2009;Segbert et al, 2004).…”

Section: Apicobasal Polarity Complexes In the C Elegans Intestinementioning

confidence: 99%

“…the CCC and DAC (Köppen et al, 2001;Kwiatkowski et al, 2010;Lockwood et al, 2008) at the future apical pole (Achilleos et al, 2010), second the assembly of a junctional belt around the apex of epithelial cells (Totong et al, 2007), third and fourth the maintenance of epithelial cell polarity Legouis et al, 2000) and cell-cell adhesion van Fürden et al, 2004).…”

Section: Apicobasal Polarity Complexes In the C Elegans Intestinementioning

confidence: 99%

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