Regulatory mechanisms of cytoneme-based morphogen transport

Abstract

During development and tissue homeostasis, cells must communicate with their neighbors to ensure coordinated responses to instructional cues. Cues such as morphogens and growth factors signal at both short and long ranges in temporal- and tissue-specific manners to guide cell fate determination, provide positional information, and to activate growth and survival responses. The precise mechanisms by which such signals traverse the extracellular environment to ensure reliable delivery to their intended cellular targets are not yet clear. One model for how this occurs suggests that specialized filopodia called cytonemes extend between signal-producing and -receiving cells to function as membrane-bound highways along which information flows. A growing body of evidence supports a crucial role for cytonemes in cell-to-cell communication. Despite this, the molecular mechanisms by which cytonemes are initiated, how they grow, and how they deliver specific signals are only starting to be revealed. Herein, we discuss recent advances toward improved understanding of cytoneme biology. We discuss similarities and differences between cytonemes and other types of cellular extensions, summarize what is known about how they originate, and discuss molecular mechanisms by which their activity may be controlled in development and tissue homeostasis. We conclude by highlighting important open questions regarding cytoneme biology, and comment on how a clear understanding of their function may provide opportunities for treating or preventing disease.

Keywords: BMP; FGF; HH; Signal transduction; Signaling filopodia; WNT.

Fig. 1

Initiation of Cytoneme Formation. A Morphogen initiation of cytoneme budding. Following maturation and secretory transport, morphogens engage cognate deployment proteins and co-receptors at the cell surface to initiate intracellular signaling through effector kinases and GTPases. Kinases activate substrates including Actin-Binding Proteins (ABPs) and Guanine nucleotide Exchange Factors (GEFs), which in turn promote GTP binding to small GTPases. GTPase effectors polymerize actin at these sites. For WNT signaling, Planar Cell Polarity (PCP) proteins contribute to deployment. Flotillin is frequently detected at sites of morphogen clustering, suggesting that it may contribute to aggregation of transmembrane and lipid-modified molecules involved in this process. B Cytoneme extension. Following activation of actin-polymerization machinery, actin is assembled into linear bundles that expand the cell surface to form a cytoneme bud. BAR domain-containing proteins are activated by Cdc42 to induce membrane curvature and concentrate actin machinery at these sites. In these cases, BAR–domain protein interactor Wiskott-Aldrich Syndrome Protein (N-WASP) promotes branched actin assembly to provide a scaffold for linear actin filament assembly. The unbranched actin polymerization, accomplished by Ena/VASP family members and/or formin proteins, then acts to extend the nascent cytoneme. Linear actin filaments are cross-linked upon binding by Fascin

Fig. 2

Morphogen Loading and Transport. A Morphogen enrichment in budding cytonemes. In polarized cells, morphogens accumulating on apical membrane are recycled in a dynamin-dependent manner for sorting to basal cytonemes. The vesicles are sorted by Rab GTPases directly to basolateral membrane for cytoneme loading through accumulating at sites of budding cytonemes or for incorporation into multi-vesicular bodies for active transport along cytoneme extensions. B Morphogen entry into existing cytonemes. Vesicles containing signaling proteins are loaded into mature cytonemes for transport to cytoneme tips by Myosin-10 (MYO10)

Fig. 3

Stabilization of Cytoneme Contacts. A Cytoneme stabilization through additive contact. Stability can be conferred by protein–protein interactions in trans. Cytoneme-localized transmembrane proteins can bind heparan sulfate proteoglycans (HSPGs) (green). For Hh (dark purple), HSPGs contribute to Ihog (blue) interactions in trans to stabilize cytoneme-cytoneme contacts (left). HSPGs in the ECM or on cytoneme membrane can also cooperate in the higher affinity Ihog–Hh interaction (center). Highest affinity interactions are achieved between the Ihog, ligand, and Hh receptor Ptc (orange) to allow for morphogen transfer and pathway activation. B Stability of cytoneme contact through CAMs. HH co-receptors BOC/Boi and CDON/Ihog also function as CAMs to stabilize these points of interaction. Dpp transport is supported by wing disc expression of CAM Capricious (Caps)/Tartan (Trn) and by ASP expression of neuronal CAMs Neuroligin 2 (Ngl2) and Neuroglian (Nrg) at cytoneme tips

Fig. 4

Morphogen Reception by Target Cells. Cytonemes can deliver signal through contact with receiving-cell bodies (A,B) or other cytonemes (C). A Signal-containing cytonemes directly contact the cell body. B Cytonemes extend into plasma membrane invaginations on the cell body. C Cytoneme tips contact to form a morphogenetic synapse where signals are presented through: (I) Incorporation into the plasma membrane at the cytoneme tip. (II) Transport along cytonemes for release from the tip. (III) Vesicular transport along the cytoneme for exovesicle-based release at a morphogenetic synapse for signaling

Fig. 5

Glutamatergic Signaling at Morphogenetic Synapses. Cytonemes from a signal-producing cell are primed for signaling through uptake of glutamate molecules (green circles) through the Vesicular Glutamate Transporter (VGlut). At the cytoneme membrane, Ca²⁺ ions (blue circles) are imported by the Voltage-Gated Calcium Channel (VGCC) and K⁺ ions (orange circles) are exported by Inward-Rectifying K⁺ Channel (Irk2) to establish an ion gradient. The Ca²⁺-binding protein Synaptotagmin 1 (Syt1) targets signal-containing vesicles to the plasma membrane for vesicle docking by Ca²⁺-dependent R-SNARE family members. Release of vesicular contents (signals and/or exosomes) at the synapse results in release of glutamate into this site. Glutamate binds the non-NMDA ionotropic glutamate receptor (GluRII) to promote its activity for Ca²⁺ uptake by the signal-receiving cell. Ca²⁺-binding Synaptotagmin 4 (Syt4) functions on the extracellular surface of the signal-receiving cytoneme to facilitate signal reception. The neuronal synaptic adhesion protein Neuroligin 2 (Nlg2) and neuronal CAM Neuroglian (Nrg) function on the ASP to stabilize this interaction