Nanotube‐like processes facilitate material transfer between photoreceptors
2021; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.15252/embr.202153732
ISSN1469-3178
AutoresAikaterini A. Kalargyrou, Mark Basche, Aura Hare, Emma L. West, Alexander J. Smith, Robin R. Ali, R. A. Pearson,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle8 September 2021Open Access Transparent process Nanotube-like processes facilitate material transfer between photoreceptors Aikaterini A Kalargyrou Corresponding Author Aikaterini A Kalargyrou [email protected] orcid.org/0000-0001-9818-030X University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Mark Basche Mark Basche University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Aura Hare Aura Hare University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Emma L West Emma L West University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Alexander J Smith Alexander J Smith University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Robin R Ali Robin R Ali University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Kellogg Eye Center, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Rachael A Pearson Corresponding Author Rachael A Pearson [email protected] orcid.org/0000-0002-1107-1969 University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Aikaterini A Kalargyrou Corresponding Author Aikaterini A Kalargyrou [email protected] orcid.org/0000-0001-9818-030X University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Mark Basche Mark Basche University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Aura Hare Aura Hare University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Emma L West Emma L West University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Alexander J Smith Alexander J Smith University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Robin R Ali Robin R Ali University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Kellogg Eye Center, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Rachael A Pearson Corresponding Author Rachael A Pearson [email protected] orcid.org/0000-0002-1107-1969 University College London Institute of Ophthalmology, London, UK Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK Search for more papers by this author Author Information Aikaterini A Kalargyrou *,1,2, Mark Basche1,2, Aura Hare1,2, Emma L West1,2, Alexander J Smith1,2, Robin R Ali1,2,3 and Rachael A Pearson *,1,2 1University College London Institute of Ophthalmology, London, UK 2Centre for Cell and Gene Therapy, King's College London, Guy's Hospital, London, UK 3Kellogg Eye Center, University of Michigan, Ann Arbor, MI, USA *Corresponding author. Tel: +44 20 7836 5454; E-mail: [email protected] *Corresponding author (lead contact). Tel: +44 20 7836 5454; E-mail: [email protected] EMBO Reports (2021)22:e53732https://doi.org/10.15252/embr.202153732 Correction added on 20 November 2021, after first online publication: The copyright line was changed. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non-neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons ("material transfer") was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open-end NT-like processes. These processes permit the transfer of cytoplasmic and membrane-bound molecules in culture and after transplantation and can mediate gain-of-function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT-like processes forming between sensory neurons in culture and in vivo. Synopsis Photoreceptors form type I and type II nanotubes (PhNTs) that mediate the transfer of cytoplasmic proteins and lipid-bound molecules and, rarely, organelles, to other photoreceptors. PhNTs mediate material transfer during photoreceptor transplantation in an actin dependent manner. Chimeric retinae reveal material transfer occurring between photoreceptors in vivo. Photoreceptors can release EVs containing photoreceptor-specific cargo in culture, which show preferential uptake by Muller Glia in culture and in vivo, but these do not mediate material transfer. Mammalian photoreceptor neurons form open-end nanotubes that mediate the exchange of cytoplasmic and membrane-bound molecules, or material transfer, which can result in gain of function in the acceptor cells. Introduction Intercellular communication is an essential process for the development and maintenance of all tissues, including the nervous system. Typically, cells employ two ways of communication, either via contacting directly (e.g. synaptic transmission) or by releasing molecular information in the extracellular fluid. Recently, new mechanisms of molecular exchange between cells have been described and include, respectively, the formation of membranous tubes between cells, called nanotubes (NTs), and the release and uptake of extracellular vesicles (EVs) (Rajendran et al, 2014; Cordero Cervantes & Zurzolo, 2021; Ljubojevic et al, 2021). Previously regarded as part of the cell's 'garbage disposal system', EVs can carry cytosolic and membrane proteins and potentially even genetic material, which have been reported to alter acceptor cell function in culture and in vivo (Kowal et al, 2016; Pastuzyn et al, 2018; van Niel et al, 2018). EVs are released by almost every cell type (van Niel et al, 2018), including neurons and glia (Faure et al, 2006; Kramer-Albers et al, 2007; Chivet et al, 2013; Ibanez et al, 2019), and may originate from the endosomal pathway or by simply budding off the plasma membrane (Kowal et al, 2016; Verweij et al, 2018). Similarly, a variety of membranous processes have been described in diverse organisms, including echinoids (where they have been termed as specialized filopodia) (Gustafson & Wolpert, 1967), flies (cytonemes) (Ramirez-Weber & Kornberg, 1999), birds (cytoplasmic bridges) (Teddy & Kulesa, 2004; George et al, 2016) and mammals (nanotubes) (Rustom et al, 2004; Chinnery et al, 2008). These processes can facilitate the exchange of molecules in culture and in vivo during early embryo development (for reviews, see (Korenkova et al, 2020; Ljubojevic et al, 2021)). Existing either as actin-enriched open-end tubes or closed-tip filopodia-like protrusions, these processes have been reported to transfer Ca2+ (Alarcon-Martinez et al, 2020), morphogens (Chen et al, 2017), fluorescent reporters (Kulesa et al, 2010; McKinney & Kulesa, 2011), vesicles (Gradilla et al, 2014), mRNA (Haimovich et al, 2017) and even organelles (Rustom et al, 2004; Alarcon-Martinez et al, 2020) between cells. Investigations into NT function in mammals have largely been limited to in vitro studies, mostly due to the technical challenges associated with their visualization. These have led to postulated roles in many pathological conditions, including viral infection, cancer, neuropathies and prion-associated disease (Gerdes & Carvalho, 2008; Gousset et al, 2009; Gerdes et al, 2013; Peralta et al, 2013; Tardivel et al, 2016). However, to our knowledge, there is only one, very recent, study reporting the presence of mammalian NT-like processes in vivo, which were shown to form between retinal pericytes and mediate the exchange of Ca2+ signals and coordinate vascular contraction (Alarcon-Martinez et al, 2020). While there are in vitro reports of NT-mediated coupling from astrocytes to neurons (Wang et al, 2012b) and within mammalian neuronal cell lines (Sun et al, 2012; Tardivel et al, 2016), it is still not known whether similar structures can form between neurons in vivo. Neuronal replacement by transplantation is proposed as a treatment for several neurodegenerative disorders. Previous studies, by us and others, have demonstrated the rescue of visual function following the transplantation of healthy photoreceptors into animal models of retinal disease (MacLaren et al, 2006; Lamba et al, 2009; Pearson et al, 2012; Barber et al, 2013; Zhu et al, 2017; Mahato et al, 2020). In end-stage retinal disease, this rescue is achieved by donor cells forming new synaptic connections with host inner retinal neurons (Ribeiro et al, 2021). However, in partial degeneration, where some/all host photoreceptor cells remain, the transplantation of healthy donor photoreceptors into recipient eyes results in the specific and surprisingly efficient transfer of a wide array of both endogenous and transgenic molecules from donor to recipient photoreceptors in both normal and diseased retina, a phenomenon that has been termed 'material transfer' (Pearson et al, 2016; Santos-Ferreira et al, 2016; Singh et al, 2016; Ortin-Martinez et al, 2017). This extraordinary finding prompted us to determine the cellular mechanism(s) underlying this exchange and to explore whether this represents a novel mechanism of non-synaptic intercellular communication between neurons, both within the normal and the diseased (treated) nervous system. We explored two leading but currently untested hypotheses (Pearson et al, 2016; Santos-Ferreira et al, 2016; Singh et al, 2016; Ortin-Martinez et al, 2017; Nickerson et al, 2018; Gasparini et al, 2019): (i) targeted release and uptake of extracellular vesicle (EVs) by photoreceptors, and (ii) physical connections between individual photoreceptor pairs, in the form of cytoplasmic bridges. Here, we used a combination of primary cell cultures, photoreceptor transplantation and the generation of chimeric retinae to elucidate the cellular mechanisms underlying material transfer between photoreceptors in culture and in vivo. Results EVs do not mediate material transfer between photoreceptors We first explored the possibility that material transfer might be mediated by the release and uptake of EVs. We examined the ultrastructure of early postnatal wildtype retinae and found that the photoreceptors often presented with multivesicular bodies (MVBs; ˜ 500 nm diameter; 1–2 MVBs/photoreceptor at postnatal day 7) containing intraluminal vesicles (ILVs) (Fig 1A), which can be released as EVs. To study EV release and other photoreceptor–photoreceptor interactions in detail, we next established a primary culture system (see Methods and Kalargyrou et al, bioRxiv) that could support purified postnatal rod photoreceptors for many days, allowing them to extend processes and release vesicles into the surrounding media (Fig 1B). EVs were enriched from the cell culture media using the standard methodology of differential ultracentrifugation (Kowal et al, 2016; Thery et al, 2018). The 100 K pellet contained vesicles of ˜ 120 nm diameter, as determined by electron microscopy (EM) (Fig 1C) and dynamic light scattering (DLS) (Fig 1D and E) analysis, and presented with markers of the endocytic machinery, including LAMP1 (Fig 1F). The classical EV tetraspanins CD81, and CD9 were also present (Fig 1F), albeit not enriched, consistent with recent reassessments of EV composition (Jeppesen et al, 2019). Conversely, the Golgi marker GM130 was absent from the 100 K sample, confirming the lack of contamination from other organelles (Fig 1F). Figure 1. Primary photoreceptors release EVs that exert their function in Müller glial cells but not photoreceptors A. TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2 µm left, 1 µm right. B. Representative SEM microphotograph of cultured P8 Nrl.Gfp+/+ photoreceptors; arrows indicate membrane budding of processes or EVs; dashed circles denote EVs; Scale bar = 3 µm. C. Representative TEM microphotograph of 100 K EV pellet derived from Nrl.Gfp+/+ photoreceptor cultures (N = 10 independent preparations); Scale bar = 100 nm. D, E. Representative dynamic light scatter (DLS) plot of 100 K EV sample (N = 13 samples), showing (D) average intensity and (E) volume against diameter (13 DLS measurements per sample). F. Representative Dot blot of 100 K EV pellet (each dot represents three pooled EV isolations, derived from 60*106 cells; N = 8 experiments) vs cell lysate (CL) from P8 Nrl.Gfp+/+ photoreceptors. EV markers = LAMP1, CD8, CD9; Golgi marker = GM130; phototransduction markers = Recoverin, Rhodopsin, Rod α-Transducin. G. RT–qPCR analysis of 100 K P8 photoreceptor pellets for Gnat1, Rec, Rho, Crx, Cre and Gfp, relative to β-actin, comparing EVs (N = 8) against the appropriate (Nrl.Gfp+/+ or Nrl.Cre+/−) photoreceptor cell lysate (N = 3). Gnat1, Cre and Gfp mRNA were present in all relevant samples. H. Schematic representation of Cre-LoxP system to assess EV function in co-culture of Nrl.Cre+/− photoreceptors (PRs) with mTmGfloxed reporter retinal cells in non-proximity (trans-well) versus physical proximity (contact). I. Representative flow cytometry analysis of Nrl.Cre+/− and mTmGfloxed co-cultures. Samples were analysed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73−ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; one-way ANOVA, non-parametric, Kruskal–Wallis with Dunns' multiple comparisons test. J. Representative maximum intensity projection (MIP) confocal images of mTmGfloxed reporter retinal cells, (left) treated with AAV-Nrl.Cre virus (positive control), (middle) co-culture with Nrl.Cre+/− photoreceptors separated by trans-well (non-proximity) or (right) in contact; Scale bar = 20 µm; Red = myrRFP-expressing mTmGfloxed reporter, no recombination; green = myrGFP-expressing mTmGfloxed reporter, cells recombined upon acquiring Cre; blue = nuclei. N = 7 cultures. Data information: Graphs show mean ± SD. Download figure Download PowerPoint We next examined whether photoreceptor-derived EVs contained cell-type specific, functionally relevant cargo, alongside reporter molecules that would permit tracing of their transfer in vitro and in vivo. Specifically, we assessed molecules previously shown to be exchanged during material transfer following photoreceptor transplantation, including the fluorescent reporter GFP, Cre and Rod α-transducin (Pearson et al, 2016; Waldron et al, 2018). Cell cultures were established from three transgenic mouse lines: Nrl.Gfp+/−, Nrl.Cre+/− and Nrl.Cre+/− x mTmGfloxed, which express cytoplasmic cGFP, Cre and myristoylated (myr)GFP, respectively. Use of the Nrl promoter ensures that expression is specific restricted to post-mitotic rod photoreceptors in the retina (Akimoto et al, 2006). Dot blot analysis demonstrated the presence of the phototransduction-related proteins Recoverin and Rhodopsin, but not Rod α-transducin (Fig 1F). Cytoplasmic GFP, myrGFP and Cre protein were detected in the 100 K pellets derived from Nrl.Gfp+/+, Nrl.Cre+/− x mTmG (membrane-tagged GFP; Appendix Fig S1A and B) and Nrl.Cre+/− photoreceptors, respectively, but varied between samples (Fig EV1A–C). Conversely, Gnat1 (rod α-transducin), Gfp and Cre mRNA was readily detectable by qRT–PCR, while Rhodopsin, Recoverin and the photoreceptor-specific transcription factor, Crx, were not detected (Fig 1G). Together, these data demonstrate that photoreceptor-derived EVs contain cargo that differs from the composition of the cytoplasm of these cells. Click here to expand this figure. Figure EV1. Photoreceptor-derived EV subpopulations contain Cre and GFP protein A–C. Representative dot blots of GFP (A), Cre (B) and GM130 (C) expression in P8 Nrl.Cre+/−, Nrl.Gfp+/+, Nrl.Cre+/− × mTmG+/+ (myrGFP) photoreceptor-derived 100 K EV pellets, as appropriate (N = 8 experiments, each dot represents a pool from three independent EV isolations, derived from 60*106 cells from two samples). The lack of GM130 staining confirms the absence of contamination from Golgi within the EV preparations. Positive GM130 staining from whole cell lysate is shown in Fig 1. D. Representative tile-scan images following (left) subretinal and (middle) intravitreal injection of EVs (100 K fraction) derived from P8 Nrl.Cre+/− photoreceptors, compared with (right) subretinal injection of EVs (100 K fraction) derived from non-photoreceptors (Nrl.Cre+/−); red = TdTomato recombined cells; blue = nuclei; Scale bar = 100 µm (left & middle) and 50 µm (right). Download figure Download PowerPoint We next examined the potential for EV-mediated transfer by employing a Nrl.Cre+/− and mTmGfloxed reporter retinal co-culture system (Fig 1H–J) followed by confocal imaging and flow cytometry analysis. Here, rod photoreceptors were enriched from Nrl.Cre+/− mice using the cell surface marker, CD73 (Eberle et al, 2011; Lakowski et al, 2015) and co-cultured with mixed retinal cells from mTmGfloxed mice a floxed Cre reporter line that ubiquitously expresses myristoylated RFP (myrRFP), but switches expression to myrGFP ("red-to-green") upon acquisition of Cre and undergoing Cre-mediated recombination (Appendix Fig S1). No spontaneous recombination was observed in untreated mTmGfloxed-only cultures, while mTmGfloxed cultures treated with AAV-CMV.Cre virus exhibited widespread recombination (Fig 1I and J). When Nrl.Cre+/− photoreceptors were co-cultured above mTmGfloxed cells and physical proximity between the two is prevented by use of a trans-well (see schematic, Fig 1H, left; and (Zomer et al, 2016)), rare examples of recombination were seen after 21 days in culture (DIC) (Fig 1I and J). The majority of recombined cells were CD73-ve (Fig 1I), indicating a non-photoreceptor identity (Eberle et al, 2011; Lakowski et al, 2011) and exhibited glial-like morphologies (Fig 1J). Others have reported that EVs may get trapped within trans-well pores (Thayanithy et al, 2017), but direct application of Nrl.Cre+/− EVs to mTmGfloxed retinal cultures also yielded very low levels of recombination, again predominantly in CD73−ve cells (Fig 1I). However, when cells were grown in physical contact with one another (see schematic, Fig 1H, right), significantly higher levels of recombination were observed. Some of these cells presented neuronal-like morphologies (Fig 1J, right) and included CD73+ve photoreceptors (Fig 1I). Together, these data indicate that cultured photoreceptors release bioactive EVs that are taken up predominantly by non-photoreceptor populations, at least in vitro. To explore this in vivo, Nrl.Cre+/− photoreceptor-derived EVs were injected into the subretinal or intravitreal space of TdTomatofloxed reporter mice ("no reporter-to-red" following Cre-mediated recombination) and compared to subretinal transplantation of Nrl.Cre+/− photoreceptors or injection of either recombinant Cre or AAV-Nrl.Cre virus (Fig 2). As we have reported previously (Pearson et al, 2016), transplantation of viable Cre+ve photoreceptors in the subretinal space results in recombination of host photoreceptors (Fig 2A and B). In notable contrast, when 100 K Nrl.Cre+/− photoreceptor-derived EVs were injected into either the subretinal space (Figs 2C and D, and 2, EV1) or intravitreally (Figs 2E and F, and EV1D) we observed striking levels of recombination but only in Müller glia cells (Fig 2D and F). Injection of the 10 K and 2 K fractions, which may additionally contain microvesicles and/or apoptotic bodies (Thery et al, 2006, 2018), also yielded Müller glia-specific labelling (Fig 2D) throughout the injection site. No other cell types were labelled. Importantly, transplantation of Nrl.Cre+/− photoreceptors that were pre-treated with UV light to induce cell death yielded no recombination (Figs 2G and EV1E), nor did subretinal injection of recombinant Cre protein (2 μg; Fig 2F, right) or non-photoreceptor-derived EVs (Fig EV1D). Transduction with AAV-Nrl.Cre virus yielded widespread recombination in the photoreceptors and some labelling of RPE cells (Fig 2B), most likely due to non-specific expression of Cre within the RPE, or possibly recombination resulting from uptake of small amounts of Cre protein. Taken together, these data demonstrate that photoreceptor cells have the capacity to release EVs, at least in culture, and if present in the intact retina, these are specifically taken up by Müller glial cells. However, EVs do not mediate material transfer between photoreceptors. Figure 2. Nrl.Cre+/− photoreceptor-derived EVs are taken up by Müller glia cells when injected in vivo A. Schematic representation of photoreceptor transplantation, shown in (B). B. (left) P8 Nrl.Cre+/− photoreceptors transplanted into subretinal space of TdTomatofloxed reporter mice (N = 5 eyes), compared to (middle) subretinal injection of AAV-Nrl.Cre subretinal injection (1012 vp/ml), (N = 10 eyes) and (right) contralateral uninjected control (N = 10 eyes). Arrows indicate recombined host photoreceptors (white arrows) or RPE (green arrows). Note clearly recombined RPE cells upon AAV transduction. C. Schematic representation of subretinal EV injection, shown in (D). Red indicates host cells undergoing Cre-mediated recombination and expressing TdTomato (red). D. P8 Nrl.Cre+/− PR -derived (left) 100 K, (middle) 10 K and (right) 2 K EV pellets injected into the subretinal space of TdTomatofloxed reporter mice (N = 4 eyes per EV preparation). Recombination was restricted to Müller Glia (asterisks). E. Schematic representation of intravitreal EV injection, shown in (F). F. (left) intravitreal injection of P8 Nrl.Cre+/− photoreceptor-derived 100 K EV pellets (N = 2 eyes). Recombination was restricted to Müller Glia (asterisks) with little or no recombination in either photoreceptors or the RPE; (right) subretinal injection of recombinant Cre protein (control) into TdTomatofloxed reporter mice. Blue = nuclei, red = TdTomato+ve recombined cells. N = 4 retinae per group. Representative MIP confocal images. G. Quantification of no. of TdTomato+ve host photoreceptor cells seen following transplantation of live (224 ± 181) versus UV-treated (dead) (3 ± 4) Nrl.Cre+/− P8 photoreceptors (N = 4 eyes per condition). Graph shows mean ± SD. Data information: Scale bars = 50 µm; PR—photoreceptor; MG—Müller Glia; RPE—retinal pigment epithelium; ONL—outer nuclear layer; INL—inner nuclear layer; GCL—Ganglion cell layer. Download figure Download PowerPoint Photoreceptors form NT-like processes in vitro In the original descriptions of material transfer in photoreceptor transplantation, donor cells were often, but not always, in close physical proximity with acceptor host cells (Pearson et al, 2016; Santos-Ferreira et al, 2016; Singh et al, 2016), sometimes apparently extending processes towards the host acceptor cells (Singh et al, 2016; Ortin-Martinez et al, 2017). We therefore considered whether physical cell–cell connections underlie photoreceptor material transfer (Fig 3). First, we performed confocal live imaging of Nrl.Gfp+/+ photoreceptor cultures labelled with the cytoskeletal probes SiR-actin or SiR-tubulin, to explore and characterize the different processes extended by photoreceptors (Fig 3A–G; Appendix Table S1; Fig EV2); these include (i) neurite-like extensions, (ii) nascent inner segment-like processes, and (iii) nanotubes. Analysis of the actin cytoskeleton revealed neurite-like extensions that terminate in actin-rich exploratory growth cones (Fig 3A), as well as thicker processes ending in bulbous inner segment-like terminals, which could be distinguished by the additional presence of tubulin (Fig 3B and E; Movie EV6). These inner segment-like processes often robustly expressed the photopigment Rhodopsin (Fig EV2A). Examining SiR-tubulin, many tubulin-rich processes were long and thick and often extended around other cells (Fig 3F). However, they only rarely appeared to form cell-to-cell connections (Fig 3G). Strikingly, however, using either probe, we also observed distinctive NT-like processes that formed continuous structures connecting the somas of pairs of adjacent cells (Fig 3C, D and G; Movies EV1–EV3). These NTs were typically short (< 10 μm), although longer lengths were observed (Fig 3C, D and G) and often remarkably straight (Fig 3H). Membrane continuity between the connected cells was further confirmed by scanning electron microscopy of Nrl.Gfp+/+ photoreceptor cultures (Fig 3H). Live imaging showed that NTs extend freely between cells and are not attached to the substratum, in contrast with neurite-like processes, which were usually attached to the substratum or other cells (Fig 3I–L); this free-floating property also makes them sensitive to fixation (Fig 3H, right). Figure 3. Photoreceptors form nanotube-like processes to connect with neighbouring photoreceptors in culture A–D. Representative 3D deconvolved MIP images from live imaging of Nrl.Gfp+/+ (green) P8 photoreceptors at DIV1–3, labelled with SiR-actin (red) (N = 7 independent cultures). Photoreceptors exhibit a variety of processes, including (A) neurites (blue arrow), (B) nascent inner segment-like protrusions (yellow arrow), and (C) short and (D) long (magenta arrows) actin+ve nanotube-like (herein termed PhNTs) connections; Scale bar = 5 µm. E–G. Representative 3D deconvolved MIP images from live imaging of Nrl.Gfp+/+ (green) P8 photoreceptors at DIV1–3 labelled with SiR-tubulin (red) (N = 7 independent cultures). Photoreceptors exhibit a variety of processes, including nascent inner segment-like protrusions (yellow arrows), neurites (blue arrows) and rare tubulin+ve PhNTs (magenta arrows); Scale bar = 5 µm. H. Representative SEM images with digitally enhanced microphotographs of cultured P8 Nrl.Gfp+/+ photoreceptors. Red arrows indicate PhNT connections between neighbouring photoreceptors, while blue arrows indicate broken PhNT connections (N = 3 cultures). Scale bar = 5 µm. I, J. Representative images of depth colour coding (ImageJ) of x,y,z live imaging of Nrl.Gfp+/+ photoreceptor cultures showing (I) neurites (blue arrows) growing along the substrate (asterisks denote secondary branching of long GFP+ neurites), and in (J), a free-floating PhNT (magenta arrow). Scale bar = 10 µm. K, L. Quantification of the localization of Nrl.Gfp+/+ processes in the z-axis (depth) (N = 4 independent cultures, n = 1,096 cells, nneurites = 440, nnanotubes = 31) where (K) neurites. P-values = ****P < 0.0001 (bottom vs top); **P = 0.002 (bottom vs middle); **P = 0.003 (middle vs top) and (L) PhNTs; P-values = ****P < 0.0001 (bottom vs middle & middle vs top). One-way ANOVA, non-parametric, Kruskal–Wallis with Dunns' multiple comparisons test shows means vary significantly. M, N. Diameter and length quantification of SiR-actin+ve PhNTs (N = 7 cultures; ncells = 646 cells; nnanotubes = 35, red dots) compared to SiR-tubulin+ve PhNTs (N = 7 cultures; ncells = 885 cells, nnanotubes = 15; blue dots). One-way ANOVA, non-parametric, Kolmogorov–Smirnov test shows non-significant (ns) difference in length but significant difference in diameter between actin+ve versus tubulin+ve PhNTs. P-values; **P = 0.001, and P = 0.245, in (M) and (N), respectively. Data information: Graphs show mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Photoreceptors form nanotube-like processes in culture A. Representative MIP example of rhodopsin immunostaining of fixed Nrl.Gfp+/+ photoreceptors at 6 DIC. Digitally enhanced microphotograp
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