Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhes... more Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays. Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated. Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component. Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b...
Nutrient-transporting enterocytes interact with their luminal environment using a densely packed ... more Nutrient-transporting enterocytes interact with their luminal environment using a densely packed collection of apical micro-villi known as the brush border. Assembly of the brush border is controlled by the intermicrovillar adhesion complex (IMAC), a protocadherin-based complex found at the tips of brush border microvilli that mediates adhesion between neighboring protru-sions. ANKS4B is known to be an essential scaffold within the IMAC, although its functional properties have not been thoroughly characterized. We report here that ANKS4B is directed to the brush border using a noncanonical apical targeting sequence that maps to a previously unannotated region of the scaffold. When expressed on its own, this sequence targeted to microvilli in the absence of any direct interaction with the other IMAC components. Sequence analysis revealed a coiled-coil motif and a putative membrane-binding basic-hydrophobic repeat sequence within this targeting region, both of which were required for the scaffold to target and mediate brush border assembly. Size-exclusion chromatography of the isolated targeting sequence coupled with in vitro brush border binding assays suggests that it functions as an oligomer. We further show that the corresponding sequence found in the closest homolog of ANKS4B, the scaffold USH1G that operates in sensory epithelia as part of the Usher complex, lacks the inherent ability to target to microvilli. This study further defines the underlying mechanism of how ANKS4B targets to the apical domain of enterocytes to drive brush border assembly and identifies a point of functional divergence between the ankyrin repeat-based scaffolds found in the IMAC and Usher complex. Intestinal enterocytes undergo an intricate morphogenic program to become specialized to mediate nutrient absorption in the gut. During terminal differentiation, each enterocyte constructs a highly ordered array of ;1000 lumen-oriented ap-ical microvilli that are enriched in nutrient processing enzymes and transport channels (1). Collectively these microvilli form the intestinal brush border (BB), a cellular adaptation that alone is responsible for nutrient uptake in the gut. Along with nutrient absorption, BB microvilli also play an important role in barrier function in the intestine. Enterocyte microvilli are coated by a thick glycocalyx at their apical tips (2, 3) and also secrete antimicrobial vesicles into the lumen of the intestine (4, 5). Together , these structural and functional properties help ensure the integrity of host tissue against potentially harmful toxins and microbes that can be found in the gut lumen. The importance of the BB is underscored by the fact that genetic conditions or acquired infections that perturb BB structure can be life-threatening, as seen with microvillus inclusion disease (6) and the attaching/effacing microbe enterohemorrhagic Esche-richia coli (7). A defining feature of the BB is its exquisite organization. During BB assembly, microvilli pack together into near-perfect hexagonal arrays, which allows for their maximum number to be constructed on the apical surface of each enterocyte (1). Furthermore , the lengths of microvilli are remarkably uniform both within and across neighboring enterocytes. Investigations into the mechanism underlying the assembly of the BB led to the discovery of a protocadherin-based adhesion complex that plays a key role in promoting BB organization (8). During construction of the BB, physical adhesion between neighboring microvilli controls their ordered packing. This adhesion is mediated by a pair of protocadherins: CDHR2 (cadherin-related family member 2; also known as protocadherin-24) and CDHR5 (cadherin-related family member 5; also known as mucin-like protocadherin) (8). These protocadherins reside at the distal tips of BB microvilli and interact in trans via a hetero-philic adhesion bond to create "intermicrovillar adhesion links" that connect neighboring microvilli to control organization. Correct targeting and function of these protocadherins require a host of cytoplasmic factors. These factors include the two scaffolding proteins USH1C (also known as Harmonin) and ANKS4B (ankyrin repeat and sterile a-motif domain containing 4B), the myosin motor protein Myo7b (Myosin-7b), and the myosin light chain CALML4 (calmodulin-like protein-4) (8-11). Altogether, these components are known as the intermi-crovillar adhesion complex (IMAC). Within the IMAC, ANKS4B forms a stable tripartite complex with USH1C and Myo7b (9, 12, 13). In both in vitro recon-stitution assays and cell-based experiments, these three IMAC components interact to form dense condensates via liquid-liquid phase separation (14). ANKS4B is comprised of an N-terminal ankyrin-repeat domain (denoted here as ANKR min), a central unstructured region (CEN), and a C-terminal sterile a-motif (SAM) ending in a PDZ-binding motif (PBM). ANKS4B uses its SAM-PBM domain to bind USH1C with high
Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhes... more Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays. Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated. Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component. Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b. Moreover, distal tip enrichment of CALML4 is strictly dependent upon its association with myosin-7b, with CALML4 acting as a light chain for this myosin. We further show that genetic disruption of CALML4 within enterocytes results in brush border assembly defects that mirror the loss of other IMAC components and that CALML4 can also associate with the Usher complex component myosin-7a. Our study further defines the molecular composition and protein-protein interaction network of the IMAC and Usher complex and may also shed light on the etiology of the sensory disorder USH1H.
Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhes... more Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays. Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated. Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component. Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b...
Nutrient-transporting enterocytes interact with their luminal environment using a densely packed ... more Nutrient-transporting enterocytes interact with their luminal environment using a densely packed collection of apical micro-villi known as the brush border. Assembly of the brush border is controlled by the intermicrovillar adhesion complex (IMAC), a protocadherin-based complex found at the tips of brush border microvilli that mediates adhesion between neighboring protru-sions. ANKS4B is known to be an essential scaffold within the IMAC, although its functional properties have not been thoroughly characterized. We report here that ANKS4B is directed to the brush border using a noncanonical apical targeting sequence that maps to a previously unannotated region of the scaffold. When expressed on its own, this sequence targeted to microvilli in the absence of any direct interaction with the other IMAC components. Sequence analysis revealed a coiled-coil motif and a putative membrane-binding basic-hydrophobic repeat sequence within this targeting region, both of which were required for the scaffold to target and mediate brush border assembly. Size-exclusion chromatography of the isolated targeting sequence coupled with in vitro brush border binding assays suggests that it functions as an oligomer. We further show that the corresponding sequence found in the closest homolog of ANKS4B, the scaffold USH1G that operates in sensory epithelia as part of the Usher complex, lacks the inherent ability to target to microvilli. This study further defines the underlying mechanism of how ANKS4B targets to the apical domain of enterocytes to drive brush border assembly and identifies a point of functional divergence between the ankyrin repeat-based scaffolds found in the IMAC and Usher complex. Intestinal enterocytes undergo an intricate morphogenic program to become specialized to mediate nutrient absorption in the gut. During terminal differentiation, each enterocyte constructs a highly ordered array of ;1000 lumen-oriented ap-ical microvilli that are enriched in nutrient processing enzymes and transport channels (1). Collectively these microvilli form the intestinal brush border (BB), a cellular adaptation that alone is responsible for nutrient uptake in the gut. Along with nutrient absorption, BB microvilli also play an important role in barrier function in the intestine. Enterocyte microvilli are coated by a thick glycocalyx at their apical tips (2, 3) and also secrete antimicrobial vesicles into the lumen of the intestine (4, 5). Together , these structural and functional properties help ensure the integrity of host tissue against potentially harmful toxins and microbes that can be found in the gut lumen. The importance of the BB is underscored by the fact that genetic conditions or acquired infections that perturb BB structure can be life-threatening, as seen with microvillus inclusion disease (6) and the attaching/effacing microbe enterohemorrhagic Esche-richia coli (7). A defining feature of the BB is its exquisite organization. During BB assembly, microvilli pack together into near-perfect hexagonal arrays, which allows for their maximum number to be constructed on the apical surface of each enterocyte (1). Furthermore , the lengths of microvilli are remarkably uniform both within and across neighboring enterocytes. Investigations into the mechanism underlying the assembly of the BB led to the discovery of a protocadherin-based adhesion complex that plays a key role in promoting BB organization (8). During construction of the BB, physical adhesion between neighboring microvilli controls their ordered packing. This adhesion is mediated by a pair of protocadherins: CDHR2 (cadherin-related family member 2; also known as protocadherin-24) and CDHR5 (cadherin-related family member 5; also known as mucin-like protocadherin) (8). These protocadherins reside at the distal tips of BB microvilli and interact in trans via a hetero-philic adhesion bond to create "intermicrovillar adhesion links" that connect neighboring microvilli to control organization. Correct targeting and function of these protocadherins require a host of cytoplasmic factors. These factors include the two scaffolding proteins USH1C (also known as Harmonin) and ANKS4B (ankyrin repeat and sterile a-motif domain containing 4B), the myosin motor protein Myo7b (Myosin-7b), and the myosin light chain CALML4 (calmodulin-like protein-4) (8-11). Altogether, these components are known as the intermi-crovillar adhesion complex (IMAC). Within the IMAC, ANKS4B forms a stable tripartite complex with USH1C and Myo7b (9, 12, 13). In both in vitro recon-stitution assays and cell-based experiments, these three IMAC components interact to form dense condensates via liquid-liquid phase separation (14). ANKS4B is comprised of an N-terminal ankyrin-repeat domain (denoted here as ANKR min), a central unstructured region (CEN), and a C-terminal sterile a-motif (SAM) ending in a PDZ-binding motif (PBM). ANKS4B uses its SAM-PBM domain to bind USH1C with high
Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhes... more Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays. Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated. Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component. Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b. Moreover, distal tip enrichment of CALML4 is strictly dependent upon its association with myosin-7b, with CALML4 acting as a light chain for this myosin. We further show that genetic disruption of CALML4 within enterocytes results in brush border assembly defects that mirror the loss of other IMAC components and that CALML4 can also associate with the Usher complex component myosin-7a. Our study further defines the molecular composition and protein-protein interaction network of the IMAC and Usher complex and may also shed light on the etiology of the sensory disorder USH1H.
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