The Dilute domain in Canoe is not essential for linking cell junctions to the cytoskeleton but supports morphogenesis robustness.

Robust linkage between adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The Drosophila multidomain protein Canoe and its mammalian homolog afadin are crucial for this, as in their absence many events of morphogenesis fail. To define the mechanism of action for Canoe, we are taking it apart. Canoe has five folded protein domains and a long intrinsically disordered region. The largest is the Dilute domain, which is shared by Canoe and myosin V. To define the roles of this domain in Canoe, we combined biochemical, genetic and cell biological assays. AlphaFold was used to predict its structure, providing similarities and contrasts with Myosin V. Biochemical data suggested one potential shared function - the ability to dimerize. We generated Canoe mutants with the Dilute domain deleted (CnoΔDIL). Surprisingly, they were viable and fertile. CnoΔDIL localized to adherens junctions and was enriched at junctions under tension. However, when its dose was reduced, CnoΔDIL did not provide fully wild-type function. Furthermore, canoeΔDIL mutants had defects in the orchestrated cell rearrangements of eye development. This reveals the robustness of junction-cytoskeletal connections during morphogenesis and highlights the power of natural selection to maintain protein structure.

The Dilute domain of Canoe is not essential for Canoe's role in linking adherens junctions to the cytoskeleton but contributes to robustness of morphogenesis.

Robust linkage between cell-cell adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The multidomain protein Drosophila Canoe and its mammalian homolog Afadin are critical for this linkage, and in their absence many events of morphogenesis fail. To define underlying mechanisms, we are taking Canoe apart, using Drosophila as our model. Canoe and Afadin share five folded protein domains, followed by a large intrinsically disordered region. The largest of these folded domains is the Dilute domain, which is found in Canoe/Afadin, their paralogs, and members of the MyosinV family. To define the roles of Canoe's Dilute domain we have combined biochemical, genetic and cell biological assays. Use of the AlphaFold tools revealed the predicted structure of the Canoe/Afadin Dilute domain, providing similarities and contrasts with that of MyosinV. Our biochemical data suggest one potential shared function: the ability to dimerize. We next generated Drosophila mutants with the Dilute domain cleanly deleted. Surprisingly, these mutants are viable and fertile, and CanoeΔDIL protein localizes to adherens junctions and is enriched at junctions under tension. However, when we reduce the dose of CanoeΔDIL protein in a sensitized assay, it becomes clear it does not provide full wildtype function. Further, canoeΔDIL mutants have defects in pupal eye development, another process that requires orchestrated cell rearrangements. Together, these data reveal the robustness in AJ-cytoskeletal connections during multiple embryonic and postembryonic events, and the power of natural selection to maintain protein structure even in robust systems.

Exploring the evolution and function of Canoe's intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis.

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function.

Exploring the evolution and function of Canoe’s intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis.

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including its C-terminal conserved motifs, are important for protein function. These data provide the foundation for further analysis of IDR function.

Performance comparison of Yb:YAG ceramics and crystal gain material in a large-area, high-energy, high average-power diode-pumped laser.

We compare for the first time the influence of different Yb:YAG gain media on the performance of a large-area, high average-power laser system with an output energy of up to 6 J. Monocrystalline slabs grown by a new technique without central growth defect are compared with ceramics. Small signal gain, maximum output energy and thermal lensing are compared for ceramic slabs with co-sintered amplified spontaneous emission (ASE) absorber cladding, monocrystalline slab with and without optically bonded ASE absorber cladding, and surface structured monocrystalline slabs. We show that these large monocrystals with optically bonded absorber cladding have similar performance to cladded ceramics, so far the only material for high-energy Yb:YAG lasers.

Developmental changes in the laminar termination of A fibre cutaneous sensory afferents in the rat spinal cord dorsal horn.

In order to establish the specificity of growth and termination of dorsal root afferents within the developing spinal cord, the central dorsal horn terminals of myelinated sensory afferents were labelled at various stages in the rat from embryonic day (E)18 through to postnatal day (P) 35 using horseradish peroxidase conjugated to choleragenoid (B-HRP). The preferential labelling of A fibre afferents with this tracer was found to be as clear in the neonate as has been reported for the adult. The results show that while the somatotopic arrangement of A fibre afferent terminals in the dorsal horn is established early in development, the laminar projections are not. Following peripheral nerve or local skin injections of B-HRP, A fibre terminals were found to project throughout laminae I to V, including lamina II (substantia gelatinosa). This widespread termination was observed consistently until the end of the third postnatal week. After P22 the terminal field becomes restricted to the normal laminae III to V.

Caring for the elderly. A new generation of patients.

As the geriatric population grows, so must our knowledge of the physiological and psychological needs unique to the elderly. This article focuses on common scenarios involving geriatric patients, such as drug-related emergencies, thermoregulatory dysfunction and mental syndromes.