Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins. In
Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition.
- Award ID(s):
- 1914542
- PAR ID:
- 10225608
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Drosophila , the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth, and cell survival. However, the upstream regulatory mechanisms of Dprs and DIPs are not clear. On the other hand, while transcription factors have been implicated in target recognition, their downstream cell surface proteins remain mostly unknown. We conduct an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We identifyhuckebein (hkb ), a transcription factor previously implicated in target recognition of the dorsal Is motor neuron. We show thathkb genetically interacts withDIP-α and loss ofhkb leads to complete removal ofDIP-α expression specifically in dorsal Is motor neurons. We then confirm that this specificity is through the dorsal Is motor neuron specific transcription factor,even-skipped (eve ), which acts downstream ofhkb . Analysis of the genetic interaction betweenhkb andeve reveals that they act in the same pathway to regulate dorsal Is motor neuron connectivity. Our study provides insight into the transcriptional regulation ofDIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons. -
ABSTRACT In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell-surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. Here, we investigated expression of Dpr and DIP genes, two CSP subfamilies belonging to the immunoglobulin superfamily, in Drosophila larval motor neurons (MNs), muscles, glia and sensory neurons (SNs) using a collection of GAL4 driver lines. We found that Dpr genes are more broadly expressed than DIP genes in MNs and SNs, and each examined neuron expresses a unique combination of Dpr and DIP genes. Interestingly, many Dpr and DIP genes are not robustly expressed, but are found instead in gradient and temporal expression patterns. In addition, the unique expression patterns of Dpr and DIP genes revealed three uncharacterized MNs. This study sets the stage for exploring the functions of Dpr and DIP genes in Drosophila MNs and SNs and provides genetic access to subsets of neurons.more » « less
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Introduction The California purple sea urchin,
Strongylocentrotus purpuratus , relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is theSpTransformer (SpTrf ) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marineVibrio . A recombinant (r)SpTrf-E1-Ec protein produced byE. coli also bindsVibrio but does not augment phagocytosis.Methods To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells.
Results The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis.
Conclusion These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.
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Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in
cis , leading to inhibitory signaling. Specifically, we construct acis -binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, thesecis -binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity. -
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Abstract Macromolecular assembly depends on tightly regulated pairwise binding interactions that are selectively favored at assembly sites while being disfavored in the soluble phase. This selective control can arise due to molecular density-enhanced binding, as recently found for the kinetochore scaffold protein CENP-T. When clustered, CENP-T recruits markedly more Ndc80 complexes than its monomeric counterpart, but the underlying molecular basis remains elusive. Here, we use quantitative
in vitro assays to reveal two distinct mechanisms driving this behavior. First, Ndc80 binding to CENP-T is a two-step process: initially, Ndc80 molecules rapidly associate and dissociate from disordered N-terminal binding sites on CENP-T. Over time, these sites undergo maturation, resulting in stronger Ndc80 retention. Second, we find that this maturation transition is regulated by a kinetic barrier that is sensitive to the molecular environment. In the soluble phase, binding site maturation is slow, but within CENP-T clusters, this process is markedly accelerated. Notably, the two Ndc80 binding sites in human CENP-T exhibit distinct maturation rates and environmental sensitivities, which correlate with their different amino-acid content and predicted binding conformations. This clustering-induced maturation is evident in dividing human cells, suggesting a distinct regulatory entry point for controlling kinetochore assembly. We propose that the tunable acceleration of binding site maturation by molecular crowding may represent a general mechanism for promoting the formation of macromolecular structures.Significance Statement A distinctive mechanism of protein-protein interaction underpins the assembly of kinetochores, which is critical for human cell division. During mitosis, the Ndc80 complex must bind tightly to the unstructured N-terminus of its receptor, CENP-T, which is densely clustered at kinetochores. Using single-molecule
in vitro assays, we show that Ndc80 binding is mediated by an initially unstable yet tunable interface. The high molecular density of CENP-T at the kinetochores accelerates the maturation of this binding interface, favoring the formation of stable complexes within the kinetochore structure, rather than in the soluble phase. This environment-driven modulation of binding site maturation may represent a key regulatory mechanism for ensuring strong and specific interactions during the assembly of macromolecular complexes such as kinetochores.
