Extracellular vesicles (EVs) are crucial for transferring bioactive materials between cells and play vital roles in both health and diseases. Cellular protrusions, including filopodia and microvilli, are generated by the bending of the plasma membrane and are considered to be rigid structures facilitating various cellular functions, such as cell migration, adhesion, and environment sensing. Compelling evidence suggests that these protrusions are dynamic and flexible structures that can serve as sources of a new class of EVs, highlighting the unique role they play in intercellular material transfer. Cytonemes are specialized filopodia protrusions that make direct contact with neighboring cells, mediating the transfer of bioactive materials between cells through their tips. In some cases, these tips fuse with the plasma membrane of neighboring cells, creating tunneling nanotubes that directly connect the cytosols of the adjacent cells. Additionally, virus particles can be released from infected cells through small bud-like of plasma membrane protrusions. These different types of protrusions, which can transfer bioactive materials, share common protein components, including I-BAR domain-containing proteins, actin cytoskeleton, and their regulatory proteins. The dynamic and flexible nature of these protrusions highlights their importance in cellular communication and material transfer within the body, including development, cancer progression, and other diseases. 

The cellular protrusions for inter-cellular material transfer: similarities between filopodia, cytonemes, tunneling nanotubes, viruses, and extracellular vesicles

Fascin crosslinks actin filaments (F-actin) into bundles that support tubular membrane protrusions including filopodia, stereocilia, and microvilli. Fascin dysregulation drives aberrant cell migration during metastasis, and fascin inhibitors are under development as cancer therapeutics. Here, we use cryo-electron microscopy and cryo-electron tomography coupled with custom denoising to probe fascin’s F-actin crosslinking mechanisms across spatial scales. Our fascin crossbridge structure reveals an asymmetric F-actin binding conformation that is allosterically blocked by the inhibitor G2. Reconstructions of seven-filament hexagonal bundle elements and variability analysis show how structural plasticity enables fascin to bridge varied inter-filament orientations, accommodating mismatches between F-actin’s helical symmetry and bundle hexagonal packing. Tomography of many-filament bundles uncovers geometric rules underlying emergent fascin binding patterns, as well as the accumulation of unfavorable crosslinks that limit bundle size. Collectively, this work shows how fascin harnesses fine-tuned nanoscale structural dynamics to build and regulate micron-scale F-actin bundles.

Fascin structural plasticity mediates flexible actin bundle construction

Tunneling nanotubes (TNTs) are thin intercellular connections that facilitate the transport of diverse cargoes, ranging from ions to organelles. While TNT studies have predominantly been conducted in cell cultures, the existence of open-ended TNTs within live organisms remains unverified. Despite the observation of intercellular connections during embryonic development across various species, their functional role in facilitating material transfer between connected cells has not been confirmed. In this study, we performed mosaic labeling of gastrula cells in zebrafish embryos to demonstrate the coexistence of TNT-like structures alongside other cellular protrusions. These embryonic TNT-like connections exhibited a morphology similar to that of TNTs described in cell culture, appeared to have similar formation mechanisms, and could be induced by Eps8 overexpression and CK666 treatment. Most notably, we demonstrated their capability to transfer both soluble cargoes and organelles, thus confirming their open-endedness. This study demonstrates the existence of functional, open-ended TNTs in a living embryo. 

Tunneling nanotubes enable intercellular transfer in zebrafish embryos

Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extra-cellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, and morphogens can only overcome the barrier by passing through a membrane-unconfined state. Under this model, SCUBE1 promotes Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and discovered novel knobs that nature can use to tune morphogen gradient sizes across tissues and organisms.

Diffusion barriers imposed by tissue topology shape morphogen gradients

Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single-molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell–cell gaps create diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. Under this model, SCUBE1 promoted Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and identified knobs that nature can use to tune morphogen gradient sizes across tissues and organisms. 

Diffusion barriers imposed by tissue topology shape Hedgehog morphogen gradients

We define cellular pliancy as the ability of a cell to adapt and respond to stress, injury or disease. Cells with low pliancy have a lower ability to respond to stress, and as a result are more prone to death. Cells with high pliancy are much more adaptable, able to survive stressful conditions, and even be prone to re-enter the cell cycle, potentially creating a permissive state for cancer initiation. We have proposed that the purposeful organization of the genome in the nucleus contributes to cell type specific pliancy. The neural retina proves to be an ideal model to study this trait, as it contains several different cell types from one common progenitor, that span the spectrum of pliancy behavior[1]. Since the model of cellular pliancy hypothesizes that the adaptability of a cell depends on its ability to access responsive genes in times of stress, the way that specific gene organization affects cellular function needs to be fully understood. general that the structure of the nucleus a

Deconstructing the Nucleus to Elucidate Cellular Pliancy in the Retina

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function. 

Cytoneme signaling provides essential contributions to mammalian tissue patterning

Journal Article Integrative Microscopy Approaches Reveal Specialized Signaling Filopodia Promote Morphogen Gradient Formation during Mammalian Development Get access Eric T Hall, Eric T Hall Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Elizabeth R Cleverdon, Elizabeth R Cleverdon Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Miriam E Dillard, Miriam E Dillard Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Yan Zhang, Yan Zhang Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Randall Wakefield, Randall Wakefield Cell and Tissue Imaging Center, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Camenzind G Robinson, Camenzind G Robinson Cell and Tissue Imaging Center, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Shondra M Pruett-Miller, Shondra M Pruett-Miller Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United StatesCenter for Advanced Genome Engineering, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Emma Selner, Emma Selner Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Shariq S Ansari, Shariq S Ansari Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Yong-Dong Wang, Yong-Dong Wang Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar ... Show more Michael Tanes, Michael Tanes Center for In Vivo Imaging and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Young Sang Ryu, Young Sang Ryu Center for In Vivo Imaging and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Daniel Stewart, Daniel Stewart Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Amanda Johnson, Amanda Johnson Cell and Tissue Imaging Center, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Jeffrey Steinberg, Jeffrey Steinberg Center for In Vivo Imaging and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Mia Panlilio, Mia Panlilio Center for Bioimage Informatics, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Khaled Khairy, Khaled Khairy Center for Bioimage Informatics, St. Jude Children’s Research Hospital, Memphis, TN, United States Search for other works by this author on: Oxford Academic Google Scholar Stacey K Ogden Stacey K Ogden Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, United States Corresponding author: Stacey.Ogden@stjude.org Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1201–1202, https://doi.org/10.1093/micmic/ozad067.618 Published: 22 July 2023 

Integrative Microscopy Approaches Reveal Specialized Signaling Filopodia Promote Morphogen Gradient Formation during Mammalian Development.

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Deconstructing the Nucleus to Elucidate Cellular Pliancy in the Retina

Cytoneme signaling provides essential contributions to mammalian tissue patterning

Integrative Microscopy Approaches Reveal Specialized Signaling Filopodia Promote Morphogen Gradient Formation during Mammalian Development.