Monitoring Editor: Ted Salmon In interphase cells, the adenomatous polyposis coli (APC) protein a... more Monitoring Editor: Ted Salmon In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody (mAb) and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with nonAPC decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-GFP and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 siRNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, AP...
When grown on rigid substrates in confluent monolayers, MDCK and other epithelial cell lines form... more When grown on rigid substrates in confluent monolayers, MDCK and other epithelial cell lines form "dome" structures. These domes are formed by cells that lifte up from the solid support but are continuous with the monolayer (as represented in the schematic below). There is sporadic data in the literature about signalling pathways that control this behaviour and I list a
Cadherins comprise a large family of membrane glycoproteins that mediate Ca § cell adhesion durin... more Cadherins comprise a large family of membrane glycoproteins that mediate Ca § cell adhesion during development and in the adult. Although the extracellular domain controls homotypic recognition and binding between cadherins on adjacent cells, proteins that bind to the cytoplasmic domain (catenins) also regulate cell adhesivity. Assembly of the cadherin/catenin complex is temporally and spatially regulated during transport to the cell surface, and, in polarized epithelial cells, different cadherin/catenin and catenin complexes have specialized sub-cellular distributions. Changes in the levels of expression, dynamics of assembly and phosphorylation of catenins directly affect cadherin function. Taken together, catenins are emerging as important linkers in cellular processes involved in adhesion, proliferation and morphogenesis.
The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well... more The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, ct-catenin,/3-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane, ot-Catenin and/~-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas
... Catch and pull a microtubule: getting a grasp on the cortex. Viki Allan 1 & Inke S. Näthk... more ... Catch and pull a microtubule: getting a grasp on the cortex. Viki Allan 1 & Inke S. Näthke 2: Viki Allan is in the School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK; ...
Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correla... more Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correlate with chromosomal instability. Here, we show that depletion of APC from cystostatic factor (CSF) Xenopus extracts leads to a decrease in microtubule density and changes in tubulin distribution in spindles and asters formed in such extracts. Addition of full-length APC protein or a large, N-terminally truncated APC fragment to APC-depleted extracts restored normal spindle morphology and the intact microtubule-binding site of APC was necessary for this rescue. These data indicate that the APC protein plays a role in the formation of spindles that is dependent on its effect on microtubules. Spindles formed in cycled extracts were not sensitive to APC depletion. In CSF extracts, spindles predominantly formed from aster-like intermediates, whereas in cycled extracts chromatin was the major site of initial microtubule polymerization. These data suggest that APC is important for centrosomally driven spindle formation, which was confirmed by our finding that APC depletion reduced the size of asters nucleated from isolated centrosomes. We propose that lack of microtubule binding in cancer-associated mutations of APC may contribute to defects in the assembly of mitotic spindles and lead to missegregation of chromosomes.
BioEssays : news and reviews in molecular, cellular and developmental biology, 2014
Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machine... more Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machinery that controls MT dynamics remains poorly understood. MT plus-end tracking proteins (+TIPs) are a set of MT-associated proteins that dynamically track growing MT ends and are uniquely positioned to govern MT dynamics. +TIPs associate with each other in a complex array of inter- and intra-molecular interactions known as the "+TIP network." Why do so many +TIPs bind to other +TIPs? Typical answers include the ideas that these interactions localize proteins where they are needed, deliver proteins to the cortex, and/or create regulatory pathways. We propose an additional and more mechanistic hypothesis: that +TIPs bind each other to create a superstructure that promotes MT assembly by constraining the structural fluctuations of the MT tip, thus acting as a polymerization chaperone.
Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial... more Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial and sporadic colonic tumours. APC is best known for its role in regulating beta-catenin, an important mediator of cell adhesion and a transcriptional activator. However, recent studies indicate that APC has additional roles in cytoskeletal regulation. It binds to microtubules directly and indirectly. Furthermore, indirect connections between APC and the actin cytoskeleton have also been described. Here, we integrate recent information describing the association between APC and the cytoskeleton to illustrate how this multifaceted protein might link different cytoskeletal elements to each other and to cellular signaling pathways.
Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat... more Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain.
Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial ... more Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.
Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins... more Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed ct-, ~-, and qc-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/ot-catenin, and either/3-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, H-catenin, and plakoglobin cosedimented as complexes. t~-Catenin was not associated with these complexes after synthesis, but a subpopulation of a-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of C ALCIUM-dependent cell-cell adhesion proteins are morphoregulatory molecules that play important roles in the structural and functional organization of cells in tissues and organs of mulficellular organisms (Takeichi,
Monitoring Editor: Ted Salmon In interphase cells, the adenomatous polyposis coli (APC) protein a... more Monitoring Editor: Ted Salmon In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody (mAb) and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with nonAPC decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-GFP and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 siRNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, AP...
When grown on rigid substrates in confluent monolayers, MDCK and other epithelial cell lines form... more When grown on rigid substrates in confluent monolayers, MDCK and other epithelial cell lines form "dome" structures. These domes are formed by cells that lifte up from the solid support but are continuous with the monolayer (as represented in the schematic below). There is sporadic data in the literature about signalling pathways that control this behaviour and I list a
Cadherins comprise a large family of membrane glycoproteins that mediate Ca § cell adhesion durin... more Cadherins comprise a large family of membrane glycoproteins that mediate Ca § cell adhesion during development and in the adult. Although the extracellular domain controls homotypic recognition and binding between cadherins on adjacent cells, proteins that bind to the cytoplasmic domain (catenins) also regulate cell adhesivity. Assembly of the cadherin/catenin complex is temporally and spatially regulated during transport to the cell surface, and, in polarized epithelial cells, different cadherin/catenin and catenin complexes have specialized sub-cellular distributions. Changes in the levels of expression, dynamics of assembly and phosphorylation of catenins directly affect cadherin function. Taken together, catenins are emerging as important linkers in cellular processes involved in adhesion, proliferation and morphogenesis.
The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well... more The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, ct-catenin,/3-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane, ot-Catenin and/~-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas
... Catch and pull a microtubule: getting a grasp on the cortex. Viki Allan 1 & Inke S. Näthk... more ... Catch and pull a microtubule: getting a grasp on the cortex. Viki Allan 1 & Inke S. Näthke 2: Viki Allan is in the School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK; ...
Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correla... more Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correlate with chromosomal instability. Here, we show that depletion of APC from cystostatic factor (CSF) Xenopus extracts leads to a decrease in microtubule density and changes in tubulin distribution in spindles and asters formed in such extracts. Addition of full-length APC protein or a large, N-terminally truncated APC fragment to APC-depleted extracts restored normal spindle morphology and the intact microtubule-binding site of APC was necessary for this rescue. These data indicate that the APC protein plays a role in the formation of spindles that is dependent on its effect on microtubules. Spindles formed in cycled extracts were not sensitive to APC depletion. In CSF extracts, spindles predominantly formed from aster-like intermediates, whereas in cycled extracts chromatin was the major site of initial microtubule polymerization. These data suggest that APC is important for centrosomally driven spindle formation, which was confirmed by our finding that APC depletion reduced the size of asters nucleated from isolated centrosomes. We propose that lack of microtubule binding in cancer-associated mutations of APC may contribute to defects in the assembly of mitotic spindles and lead to missegregation of chromosomes.
BioEssays : news and reviews in molecular, cellular and developmental biology, 2014
Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machine... more Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machinery that controls MT dynamics remains poorly understood. MT plus-end tracking proteins (+TIPs) are a set of MT-associated proteins that dynamically track growing MT ends and are uniquely positioned to govern MT dynamics. +TIPs associate with each other in a complex array of inter- and intra-molecular interactions known as the "+TIP network." Why do so many +TIPs bind to other +TIPs? Typical answers include the ideas that these interactions localize proteins where they are needed, deliver proteins to the cortex, and/or create regulatory pathways. We propose an additional and more mechanistic hypothesis: that +TIPs bind each other to create a superstructure that promotes MT assembly by constraining the structural fluctuations of the MT tip, thus acting as a polymerization chaperone.
Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial... more Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial and sporadic colonic tumours. APC is best known for its role in regulating beta-catenin, an important mediator of cell adhesion and a transcriptional activator. However, recent studies indicate that APC has additional roles in cytoskeletal regulation. It binds to microtubules directly and indirectly. Furthermore, indirect connections between APC and the actin cytoskeleton have also been described. Here, we integrate recent information describing the association between APC and the cytoskeleton to illustrate how this multifaceted protein might link different cytoskeletal elements to each other and to cellular signaling pathways.
Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat... more Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain.
Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial ... more Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.
Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins... more Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed ct-, ~-, and qc-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/ot-catenin, and either/3-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, H-catenin, and plakoglobin cosedimented as complexes. t~-Catenin was not associated with these complexes after synthesis, but a subpopulation of a-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of C ALCIUM-dependent cell-cell adhesion proteins are morphoregulatory molecules that play important roles in the structural and functional organization of cells in tissues and organs of mulficellular organisms (Takeichi,
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