Figura 3 PDF

letters
Prions hijack tunnelling nanotubes for intercellular

spread
Karine Gousset1,7, Edwin Schiff1,2,7, Christelle Langevin1, Zrinka Marijanovic1, Anna Caputo1,3, Duncan T. Browman1,
Nicolas Chenouard4, Fabrice de Chaumont4, Angelo Martino5, Jost Enninga6, Jean-Christophe Olivo-Marin4,
Daniela Männel2 and Chiara Zurzolo1,3,8
In variant Creutzfeldt–Jakob disease, prions (PrPSc) enter the We found that CAD cells readily form membrane bridges19 that contain
body with contaminated foodstuffs and can spread from the actin and do not touch the substratum9–12 (Fig. 1a). To determine the fre-
intestinal entry site to the central nervous system (CNS) by quency of these structures and to exclude incomplete cytokinesis of daugh-
intercellular transfer from the lymphoid system to the peripheral ter cells, we co-cultured cells transfected with green fluorescent protein
nervous system (PNS)1. Although several means2–4 and different (GFP)–actin and Cherry–actin (Fig. 1b). Overall, these experiments indi-
cell types5–7 have been proposed to have a role, the mechanism cated that 44 ± 6% (s.d.) of the observed CAD cells could efficiently form
of cell-to-cell spreading remains elusive. Tunnelling nanotubes actin-containing membrane bridges of different lengths and diameters
(TNTs) have been identified between cells8–12, both in vitro and between differentially labelled populations (Fig. 1b–d; see Supplementary
in vivo10,11,13, and may represent a conserved means of cell-to- Methods). To determine whether some of these connections were tun-
cell communication14–16. Here we show that TNTs allow transfer nelling nanotubes that would permit intercellular vesicle transfer, we
of exogenous and endogenous PrPSc between infected and naive co-cultured untransfected CAD cells with GFP–actin-transfected cells.
neuronal CAD cells17. Significantly, transfer of endogenous After 24 h, the cells were labelled with LysoTracker red and imaged by
PrPSc aggregates was detected exclusively when cells chronically spinning-disk confocal microscopy (Fig. 2a). Figure 2a shows a GFP–
infected with the 139A mouse prion strain were connected to actin-transfected CAD cell (green) that has extended a tubular structure
mouse CAD cells by means of TNTs, identifying TNTs as an to a cell stained with LysoTracker (red). Over time, a LysoTracker-positive
efficient route for PrPSc spreading in neuronal cells. In addition, vesicle can be seen moving toward the GFP–actin-transfected cell and
we detected the transfer of labelled PrPSc from bone marrow- entering its cytoplasm (Fig. 2b; Supplementary Movie S1), indicating
derived dendritic cells to primary neurons connected through that this tube was in fact a functional TNT. Measurements of the mean
TNTs. Because dendritic cells can interact with peripheral neurons square displacement of vesicles demonstrate that the vesicle has a directed
in lymphoid organs, TNT-mediated intercellular transfer would movement towards the recipient cell, as opposed to random Brownian
allow neurons to transport prions retrogradely to the CNS1. We movement (Supplementary Information, Fig. S1a). The estimated velocity
therefore propose that TNTs are involved in the spreading of PrPSc of the tracked vesicle (Fig. 2c; Supplementary Movie S2) is very similar to
within neurons in the CNS and from the peripheral site of entry to that calculated for a complex of myosin VI and the GLUT1 transporter
the PNS by neuroimmune interactions with dendritic cells. binding protein20, indicating the possible involvement of actin-mediated
motors, as previously suggested for TNT vesicular transport8. Thus, in
To examine whether TNTs represent a biological method for prion CAD cells, vesicles of lysosomal origin are actively transferred intercel-
spreading in neuronal cells we used CAD cells, a mouse neuronal cell line lularly through TNTs.
of catecholaminergic origin that expresses neuron-specific proteins17. To analyse whether CAD cells could use TNTs to transfer PrPC in
These cells have been shown to be an excellent neuronal cell culture live conditions, we transfected them with GFP-tagged wild-type PrP
model because they can differentiate17 and they have also been shown (GFP–PrPwt). Interestingly, GFP–PrPwt could be seen in TNTs, includ-
to efficiently replicate prion strains18. ing in networks bridging multiple cells (Fig. 2d), both at the surface and
1
Unité de Trafic Membranaire et Pathogénèse, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. 2Department of Immunology, University of
Regensburg, F.-J.-Strauss-Allee, 93042 Regensburg, Germany. 3Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli ‘Federico
II’, via Pansini 5, 80131 Naples, Italy. 4Unité d’Analyse d’Images Quantitative, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. 5Unité de
recherché de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. 6Groupe “Dynamique des interactions hôte-pathogène”,
Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.
7
These authors contributed equally to this work.
8
Correspondence should be addressed to C.Z. (e-mail: zurzolo@unina.it; zurzolo@pasteur.fr)
Received 11 August 2008; accepted 26 January 2009; published online 8 February 2009; DOI:10.1038/ncb1841
328 nature cell biology volume 11 | number 3 | MARCH 2009

© 2009 Macmillan Publishers Limited. All rights reserved.
letters
a b 100
90
Percentage of CAD cells

80
connected by TNTs
70
60
n = 195 n = 426
50
n = 162
40 n = 69
30
20
10
0
GFP–actin DiI Cherry/ Average
GFP–actin
c d 80
40
70
35
60
Percentage of TNTs
Percentage of TNTs
30
50
25
40
20
30
15
20
10
10
5
0 0 Less than 248 459 827

13.9 25.1 39.2 74.5 186 ± 48 ± 95 ± 207
± 2.4 ± 2.7 ± 4.3 ± 8.6
Average length of TNTs (µm) Average diameter of TNTs (nm)
Figure 1 TNT analyses in mouse neuronal CAD cells. (a) Three-dimensional TNTs in CAD cells. Fifty random TNTs were analysed from three independent
reconstruction of a network of TNTs in CAD cells. The three-dimensional experiments, using CAD cells transfected with GFP–actin or labelled with DiI.
reconstruction of CAD cells labelled with DiI, showing that the tubes Percentages of cells are plotted as a function of the lengths of TNTs, showing
are not attached to the substratum, was performed with OsiriX software. that TNTs are very heterogeneous in length. (d) Diameters of TNTs in CAD
Scale bar, 10 µm. (b) High frequency of TNTs in CAD cultures. CAD cells cells. Fifty random TNTs were analysed from three independent experiments,
were transiently transfected with GFP–actin and Cherry–actin or were using CAD cells transfected with GFP–actin or labelled with DiI. Percentages
untransfected. The untransfected cells were labelled with DiI. The number of of cells are plotted as a function of the average diameters of TNTs. We found
cells counted in two independent experiments is indicated. The percentage of that most (72%) had a diameter of less than 186 nm, which is consistent
cells having one or more TNT was plotted for each condition, and the average with the previously reported diameter for TNTs in PC12 cells15,34. All images
for all three conditions was determined. Data are mean ± s.d. (c) Length of were acquired with an Andor spinning-disk confocal microscope.
in vesicular structures of lysosomal origin labelled with LysoTracker PrP (Alexa-PrPSc) by modifying a previously published protocol22
inside TNTs (Supplementary Information, Fig. S1c). These tubes were (Supplementary Information, Fig. S2a; see Supplementary Methods).
in constant movement and could mediate the transfer of GFP–PrPwt Similarly to what was shown previously in the neuronal cell line SN56
from one cell to another, possibly by surface continuity with the TNTs and primary cultured neurons22, Alexa-PrPSc is efficiently taken up
and by vesicle trafficking (Fig. 2e, Supplementary Movies S3 and S4). by CAD cells and is highly enriched in PrPSc, as shown by immun-
In this case measurements of the mean square displacement of vesicles ofluorescence with anti-PrP Sha31 antibody after denaturation with
demonstrate the directed movement of vesicles in TNTs (Supplementary guanidinium (Gnd) (Fig. 3b). In addition, it remains infectious after
Information, Fig. S1b). Furthermore, by applying a new tracking analy- labelling, as shown by our cerebellar granular neuron (CGN) infection
sis (Fig. 2f; Supplementary Movie S5 and Supplementary Methods) we assay (Fig. 3c). For a direct analysis of TNT-mediated transfer of Alexa-
determined that this vesicle was moving faster than the LysoTracker- PrPSc between the cells, CAD cells were loaded with Alexa-PrPSc, washed
labelled vesicle in Supplementary Movie S1 but remained within the range extensively and co-cultured for 24 h with wild-type CAD (Fig. 3d) or
of the velocities of actin molecular motors21, which is consistent with all CAD cells previously transfected with GFP–GPI (Fig. 3e). By immun-
of the analysed vesicles (data not shown). Overall, these data show that ofluorescence we could detect TNTs containing Alexa-PrPSc, recognized
GFP–PrPwt transfected into cells of neuronal origin can be transferred by Sha31 antibody after treatment with Gnd (Fig. 3d), and we found
through TNTs, probably by moving on the plasma membrane and by Alexa-labelled particles in the lumen of non-loaded cells (Fig. 3e, arrows
vesicular transport inside TNTs. This is consistent with previous findings and inset), suggesting transfer of PrPSc through TNTs. Analyses of mov-
showing a model glycosylphosphatidylinositol (GPI)-anchored protein ies showed Alexa-PrPSc moving within TNTs (Supplementary Movie S6;
(GFP–GPI) moving in TNTs9. details in Supplementary Movie S7). Importantly, we could not detect
Next, to determine whether PrPSc could be transferred through TNTs any fluorescence when adding the supernatant from the Alexa-PrPSc-
we co-cultured CAD cells loaded with mouse 139A brain homogenate loaded cells to unlabelled cells (data not shown), thus excluding transfer
with CAD cells transfected with GFP–GPI. PrPSc was observed in TNTs of fluorescently labelled PrPSc through the supernatant.
as well as in the cytoplasm of non-infected GFP–GPI CAD cells, sug- Taken together, our results support the hypothesis that infectious
gesting a possible transfer of scrapie through TNTs (Fig. 3a). To make PrPSc was transported from loaded cells to recipient cells through TNTs,
visible the live transfer of PrPSc, we produced a fluorescently labelled suggesting that this could be a method of spreading infection between
nature cell biology volume 11 | number 3 | MARCH 2009 329

letters
a LysoTracker GFP–actin b c
90
80
70
Velocity (nm s–1)

0s 450 s 60
50
40
30
20
150 s 600 s
10
3D reconstruction 0
5
0
0
5
5
5
5
5
5
75
75
75
37
10
47
57
67
77
87
97
1,0
1,1
1,2
Time (s)
300 s 750 s
f
350
300
0s 20 s 40 s
Velocity (nm s–1)

250
200
d
150
100
60 s 80 s 100 s
50
0
0 40 80 120 160 200
Time (s)
120 s 140 s 160 s
180 s 200 s 220 s
Figure 2 LysoTracker-labelled vesicles and GFP–PrPwt transfer through GFP–PrPwt trafficking. (d) Transfer of GFP–PrPwt vesicles through TNTs. A
TNTs between CAD cells. (a–c) LysoTracker vesicle trafficking. (a) Transfer network of TNTs between CAD cells transfected with GFP–PrPwt is shown
of LysoTracker vesicles. A mixture of untransfected cells and CAD cells (see Supplementary Movie S3) is shown. (e) Selected frames from a video
transfected with GFP–actin were labelled with LysoTracker and imaged by from the boxed area in d (see Supplementary Movies S4 and S5). Arrowheads
live microscopy. The merged fluorescence and a three-dimensional (3D) follow a GFP–PrPwt-positive vesicle moving inside a TNT and entering another
reconstruction (with OsiriX) showing a TNT (yellow arrow) between the two cell cell. (f) Velocities of a GFP–PrPwt vesicle inside a TNT. The velocity is plotted
types are shown. (b) Selected frames of a video sequence (see Supplementary as a function of time. The GFP–PrPwt vesicle moves with an average speed
Movies S1 and S2). Arrowheads highlight a LysoTracker-positive vesicle of 180 ± 83 nm s−1 in the TNT from t = 0 s to t = 50 s and slowed down
moving inside a TNT. The vesicle moves along the tube and enters the towards the end of the tube to an average speed of 23.4 ± 13.7 nm s−1 from
cytoplasm of the green recipient cell. (c) Velocity of the LysoTracker vesicle. t = 60 s to t = 190 s, before entering the cell at t = 200 s. Thus, the vesicle
The velocity of the vesicle in b is plotted as a function of time. Vesicle tracking analysed had a directed movement and was able to move at an average rate of
analysis estimated an average speed of 41.5 nm s−1 over the first 450 s. (d–f) 180 ± 83 nm s−1 within the TNT. Scale bars, 10 µm.
neuronal cells. However, although we could not find major protein strain (ScCAD) by immunofluorescence of fixed samples in cells pre-
contaminants in our Alexa-PrPSc preparations by mass spectrometry treated with Gnd (Fig. 4). As shown previously, after the Gnd treatment
(Supplementary Information, Fig. S2b, c; Supplementary Methods), it required to reveal PrPSc epitopes, the signals from PrPC and PrPSc were
remains an artificial system for the analysis of PrPSc transfer. As reported very different23 and easily recognizable in our CAD cell co-cultures
previously22, this preparation is very heterogeneous and is composed (Fig. 4a). To identify the two cell populations, non-infected CAD cells
of different particle sizes, including larger particles that cannot enter were transfected with Cherry-tagged placental alkaline phosphatase
TNTs. In addition, few particles remained in TNTs after treatment with (Cherry–PLAP) and co-cultured overnight with ScCAD cells. Using
Gnd (Fig. 3d, e), thus preventing the possibility of performing quantita- anti-PrP Sha31 antibody after Gnd treatment, we could clearly recognize
tive analyses of the transfer. Therefore, to obtain a robust quantitative endogenous PrPSc in TNTs and in the recipient non-infected CAD cells.
analysis of transfer, we decided to investigate whether endogenous PrPSc Indeed, although many TNTs were disrupted by fixation and Gnd treat-
would spread from cells chronically infected with the 139A mouse prion ment, the thickest ones remained (Supplementary Information, Fig. S3a;
strain (ScCAD cells) to non-infected CAD cells through TNTs. compare with Fig. 1d), and we found both PrPC (Fig. 4b) and PrPSc within
First we set up the conditions to analyse PrPc and PrPSc in non-infected vesicular structures inside the TNTs and in the cytoplasm of recipient cells
CAD or in CAD cells chronically infected with the 139A mouse prion (Fig. 4b, c). To quantify the transfer of endogenous PrPSc from infected

letters
a d
GFP–GPI / 139A PrPSc
GFP–GPI / 139A PrPSc
b
–Gnd +Gnd
Alexa-PrPSc / PrP/ + Gnd

e
Alexa-PrPSc / PrP GFP–GPI
c 139A PTA Alexa-PrPSc

Mr(K) Mr(K)
34 34 Alexa-PrPSc
34
2
1
17 17
17
Days: 7 Merge X-Y
14 21 7 14 21 7 14 21
Figure 3 Brain homogenate and infectious Alexa-PrPSc transfer through (139A), brain homogenate purified with PTA or Alexa-labelled PTA (Alexa-
TNTs. (a) CAD cells loaded with 139A brain homogenate transfer PrPSc PrPSc). Cell lysates from CGN cultures after 7, 14 and 21 days were treated
through TNTs. Left: cultures of CAD cells loaded with brain homogenate with PK, and PrP-res was detected by immunoblotting with Sha31. PrP-res
and GFP–GPI-transfected CAD cells were fixed, denatured with Gnd accumulation is observed over time for all conditions. Uncropped blots are
and immunolabelled with the anti-prion antibody Sha31 and AlexaFluor shown in Supplementary Information, Fig. 8a–c. (d) Alexa-PrPSc is transferred
488-conjugated secondary antibody. Cells loaded with brain homogenate through TNTs. CAD cells loaded with Alexa-PrPSc were co-cultured with non-
revealed a punctate PrPSc pattern (red). PrPSc was detected within TNTs and loaded CAD cells, fixed, denatured and immunolabelled with Sha31 and
in the cytoplasm of non-infected GFP–GPI CAD cells, suggesting transfer AlexaFluor 488-conjugated secondary antibody. Aggregates of Alexa-PrPSc, co-
through TNTs. Right: magnification of the boxed areas. (b) Immunolabelling localizing with Sha31, are present in TNTs (yellow arrows). (e) Alexa-PrPSc is
of Alexa-PrPSc with Sha31. CAD cells loaded with Alexa-PrPSc were fixed transferred through TNTs between different cells. ScCADs loaded with Alexa-
(−Gnd), denatured with Gnd (+Gnd) and immunolabelled with Sha31 and PrPSc were co-cultured with GFP–GPI-transfected CAD cells. The presence of
AlexaFluor 488-conjugated secondary antibody (green). The yellow colour red vesicles inside the TNT and in the cytoplasm of the transfected CAD cell
represents co-localization of Alexa-PrPSc and Sha31. Alexa-PrPSc does not (green) suggests a TNT-mediated transfer of Alexa-PrPSc between different
contain PrPC (−Gnd) but is highly enriched in PrPSc (+Gnd). Images are cell populations. To reveal all Alexa-PrPSc particles, high laser power was
representative of three independent experiments. (c) Alexa-PrPSc remains used, resulting in the detection of highly fluorescent Alexa-PrPSc particles in
infectious. Cerebellar granule neurons were exposed to brain homogenate the green channel. Scale bars, 10 µm.
to non-infected cells, we counted the punctate staining, representing the CAD populations into three categories: (1) non-touching CAD and
PrPSc aggregates in non-infected CAD cells after overnight co-cultures ScCAD cells, (2) direct membrane contact between CAD and ScCAD
with ScCAD cells (Supplementary Information, Fig. S3b, c). We divided cells, and (3) contact of CAD cells with ScCAD cells by means of TNTs

letters
a c
PrPC PrPC
PrPSc Cherry–PLAP Merge
PrPSc
PrP Sc
d
Percentage of CADs containing PrPSc

70
1 2
60
*
50 **
***
40
b 30 3 4
20
10
0
PrPC Cherry–PLAP Merge 1 2 3 4
Figure 4 Detection and quantification of endogenous PrPSc transfer Cherry–PLAP-transfected cell, showing that PrPSc can transfer through
in CAD and ScCAD cells through TNTs. (a) Detection of PrP in CAD TNTs. (d) Quantification of endogenous PrPSc transfer from ScCADs to CAD
cells. CAD cells (top) or ScCAD cells (bottom) were fixed, denatured cells through TNTs. Cherry–PLAP-transfected CAD cells (red) co-cultured
and labelled with Sha31 and AlexaFluor 488-conjugated secondary with ScCAD (columns 1–3) or with ScCAD in the presence of latrunculin
antibody. PrPC is mostly at the plasma membrane without bright puncta to block TNT formation (column 4) were fixed, denatured, immunolabelled
in the cells. In contrast, most PrPSc signals are very bright puncta in with Sha31 and AlexaFluor 488-conjugated secondary antibody (green),
the cytoplasm of ScCAD cells. (b) Endogenous PrPC-enriched TNTs are imaged and grouped according to the following cases: column 1, CAD
observed between cells. Cherry–PLAP-transfected CAD cells (red) co- cells not touching ScCAD cells (n = 57); column 2, CAD cells in direct
cultured with CAD cells were fixed, denatured and immunolabelled with cell contact with ScCAD cells (n = 54); column 3, CAD cells in contact
Sha31 and AlexaFluor 488-conjugated secondary antibody (green). with ScCAD cells through TNTs (n = 27); column 4, latrunculin-treated
Green TNTs between different CAD populations are visible. (c) Transfer co-cultures (n = 74). Efficient transfer of PrPSc is detected only in cells
of PrPSc through TNTs between ScCAD cells and non-infected CAD cells. connected through TNTs. Images 1–4 are representative examples for each
Cherry–PLAP-transfected CAD cells (red) co-cultured with ScCAD cells of these cases. Fischer’s exact test: asterisk, P = 1.62 × 10−6 (n = 84);
were fixed, denatured and immunolabelled with Sha31 and AlexaFluor two asterisks, P = 2.46 × 10−7 (n = 81); three asterisks, 1.60 × 10−7
488-conjugated secondary antibody (green). PrPSc was found in vesicular (n = 101). Scale bars, 10 µm. Images are representative of at least three
structures (white arrows) inside TNTs, as well as in the cytoplasm of the independent experiments.
(Fig. 4d, columns 1–3). Similarly to what we observed with exogenous endogenous PrPSc can be transferred efficiently from infected cells to
PrPSc, after overnight co-culture we could not detect PrPSc particles in recipient cells of neuronal origin.
naive cells that were not in contact with ScCAD cells (Fig. 4d, column 1; TNTs may also be produced in vivo, as shown in Drosophila10,11,14,
Supplementary Information, Fig. S3b, column 1), thus excluding exo- representing a method of communication between distant cells of dif-
somal transfer or protein shedding. Similarly, no transfer was observed ferent origins. For scrapie, TNTs could represent an efficient mechanism
between cells in direct contact with one another (Fig. 4d, column 2; and for the in vivo transfer of PrPSc by cells at the peripheral entry site to
Supplementary Information, Fig. S3b, column 2), which is in agreement neurons1. Dendritic cells (DCs), which are very apt at producing TNTs
with previous data obtained with SN56 cells22, where transfer between both in vitro24 and in vivo13, can sample and transport PrPSc from the
contacting cells was reported to be a rare event22. Strikingly, the detec- gut to peripheral lymphatic organs and are sufficient for de novo infec-
tion of PrPSc aggregates in non-infected CAD cells occurred only in cases tion when adoptively transferred from infected to healthy animals1,5,25.
where TNTs were present between the donor ScCAD cells and the recipi- Lymphatic organs such as the spleen are innervated by the sympathetic
ent CAD cells (Fig. 4d, column 3; Supplementary Information, Fig. S3b, nervous system, which in infected animals has been shown to contain
column 3, Fig. S3c; Supplementary Methods). As an additional control, PrPSc in abundance26. Intriguingly, DCs are found in close proximity
we used the actin-depolymerizing agent latrunculin to inhibit TNT for- to the PNS27,28, and their involvement in prion transfer to the PNS has
mation8. When used overnight at 100 nM concentration, both CAD and been suggested1,5,25,29.
ScCAD cells rounded up and were no longer able to make projections, We therefore analysed whether TNTs can mediate PrPSc transfer
including filopodia, membrane ruffles or TNTs. Under these conditions, between DCs and neurons. By immunohistofluorescence on splenic
no transfer of PrPSc was observed, regardless of whether the CAD and sections we found DCs closely juxtaposed to neurites (Supplementary
ScCAD cells were touching or not (Fig. 4d, column 4; Supplementary Information, Fig. S4) of the sympathetic nervous system emanating
Information, Fig. S3b, column 4). The detection of PrPSc in non-infected from the central arteriole (Supplementary Information, Fig. S4, aster-
CAD cells was therefore restricted to cases where TNTs were present isk). Because present imaging systems do not have sufficient resolution
between the donor and recipient cells, resulting in 45% of the Cherry– to analyse TNTs in spleen tissue, we first used differentiated CAD cells
PLAP non-infected CAD cells containing PrPSc inside their cytoplasm produced by serum starvation, as described previously17, and exam-
(Fig. 4d). Overall, our results indicate that TNTs are one route by which ined whether they could interact with bone-marrow-derived DCs

letters
a b c
DC
DIC DIC
GFP–PrPwt GFP–PrPwt
d
DC
NI 139A co-culture
Mr(K)
LysoTracker Alexa-PrPSc
34
17
Days: 14 14 21 14 21 Merge Merge
Figure 5 BMDCs can interact with primary neurons through TNTs to spread with a neurite by means of TNTs (arrowheads). Several Alexa-PrPSc particles
infection. (a) TNT-mediated transport of Alexa-PrPSc between BMDCs and are observed (arrows): at the base of the TNTs, inside the neurite, and several
differentiated CAD cells. Sequential pictures show the movement of Alexa- in the cell body of the neuron (arrows). Scale bars, 5 µm. (d) Infection of
PrPSc (yellow arrow) within a TNT between a loaded BMDC and the axon of cerebellar granule neurons (CGN) in co-cultures with BMDCs loaded with
a differentiated CAD cell. (b) Transport of lysosomal vesicles through TNTs 139A brain homogenate. PrP-res analysis after digestion with PK was
between BMDCs and hippocampal neurons. LysoTracker-labelled BMDCs performed on CGN cultures at 14 or 21 days after co-culture with BMDCs
(red) co-cultured with primary hippocampal neurons transduced with GFP– loaded with brain homogenate (DC co-culture) or after direct exposure to
PrPwt (green) interact by means of TNTs (arrowhead) and transfer lysosomal brain homogenate (139A). In both cases, PrP-res accumulation is observed
vesicles (arrows). DIC, differential interference contrast. (c) Transport of in CGN cultures after 21 days. No signal could be detected in non-infected
Alexa-PrPSc through TNTs between BMDCs and hippocampal neurons. In cells (NI). Immunoblot was performed with Sha31, and uncropped blots are
a co-culture of Alexa-PrPSc-loaded BMDCs and GFP–PrPwt-transduced shown in Supplementary Information, Fig. 8d. Gels are representative of
hippocampal neurons, a BMDC containing Alexa-PrPSc is shown interacting three independent experiments.
(BMDCs). In overnight co-cultures, we observed TNTs between the We then determined whether BMDCs could also interact with pri-
two cell types (Supplementary Information, Fig. S5a; Supplementary mary neurons. We noticed numerous TNTs in overnight co-cultures
Movies S8 and S9) and witnessed the transfer of Alexa-PrPSc from between BMDCs and CGNs (Supplementary Information, Fig. S5b;
a loaded BMDC to the axon of a differentiated CAD cell (Fig. 5a). Supplementary Movie S12) and between BMDCs and primary hippoc-
Furthermore, time-lapse analyses of movies obtained with another ampal neurons (Supplementary Movie S13). In addition, TNTs were seen
imaging system (Nikon Biostation) over a period of 24 h demon- to connect GFP–PrPwt-transduced hippocampal neurons and BMDCs
strated the formation of numerous TNTs, both between CAD cells (stained in red with LysoTracker) (Fig. 5b). The presence of LysoTracker-
and between BMDCs and CAD cells as early as 30 min after addition of stained vesicles inside neurites connected to BMDCs by TNTs suggests
the BMDCs to the differentiated CAD cells. With this imaging system, the transfer of LysoTracker-positive vesicles from BMDCs to neurons
intact TNTs could be observed for extended periods (more than 1 h) (Fig. 5b). To determine whether PrPSc is also transferred between these
between cells (Supplementary Movies S10 and S11), further suggest- two cell types through TNTs, BMDCs were loaded with Alexa-PrPSc and
ing that TNTs could provide an efficient means of communication co-cultured with GFP–PrPwt-transduced hippocampal neurons. Under
between distant cells. these conditions we found TNTs connecting the two different cell types

letters
(Fig. 5c, arrowheads) and we observed Alexa-PrPSc in the TNT itself, Methods

in the neuronal cell bodies, and in the neurites close to the intercellular Cell lines, antibodies, transfections and transductions. CAD cells (mouse cat-
connection (Fig. 5c, arrows; Supplementary Information, Fig. S6). These echolaminergic neuronal cell line, Cath.a-Differentiated) and cells chronically
infected with prion strain 139A (ScCAD), gifts from H. Laude, were cultured in
data show that BMDCs can interact with neurons by means of TNTs and Opti-MEM (Invitrogen) and 10% fetal bovine serum. moRK13 cells were a gift from
suggest that transfer of infectious prions between these two cell types Andrew F. Hill (University of Melbourne). Transient transfections were performed
might occur through these structures. with Lipofectamine (Invitrogen) in accordance with the manufacter’s instructions.
To determine the biological relevance of BMDCs in prion transfer, we pEGFP–C1 (Clontech) was engineered to express mouse PrPC between the NheI
analysed whether BMDCs could directly spread infection to neurons. and EcoRI restriction sites, and the signal peptide of mouse PrPC was inserted 5´
of enhanced GFP (EGFP) between the restriction sites of NheI and PinaAI (gift
Because it has been reported30 that DCs can degrade PrPSc, we loaded
from M. Sorgato, University of Padua). The GFP–actin construct was a gift from
BMDCs with 139A brain homogenate (Supplementary Information, the laboratory of P. Cossart (Pasteur Institute, Paris). Cherry–actin was a gift from
Fig. S7a) and analysed the degradation of PrP-res over time (Supplementary Michael Way (Cancer Research Institute, London). Murine wild-type PrP was
Information, Fig. S7b). Because the levels of PrP-res decrease drastically at inserted in the vector pRRLsin.PPT.hPGK.GFPpre (gift from J. M. Heard, Institut
72 h after loading (Supplementary Information, Fig. S7b), we co-cultured Pasteur, Paris) using the SalI and BamHI restriction sites, at a position 3´ of GFP.
The lentiviral vector was produced and collected as described elsewhere35. The
loaded BMDCs with primary neurons for just 12 h and, after removing
anti-PrP antibody Sha31 (residues 148–155, human numbering) was purchased
BMDCs, analysed PrP-res formation in neurons at two and three weeks after from Spibio; AlexaFluor 546 carboxylic acid succinimidyl ester and AlexaFluor
co-culturing. We observed a substantial increase in PrPSc levels in neuronal 488-conjugated secondary antibody were purchased from Invitrogen.
cultures within three weeks (Fig. 5d). In contrast, neurons were not infected
Primary cell cultures. Primary cultures of CGNs, BMDCs and embryonic hip-
if exposed for the same time to the supernatant of infected BMDCs (data
pocampal neurons were produced as described previously36–38.
not shown). These data strongly suggest that PrPSc was efficiently trans-
ferred from BMDCs to neurons and that new PrPSc synthesis took place in Loading of BMDCs with infected brain homogenate. 139A brain homoge-
the neurons. Furthermore, because of our short co-culturing time, infection nate (5 mg), prepared as described previously36 from terminally ill infected mice,
was sonicated in Iscove’s modified Dulbecco’s medium (IMDM; BioWhittaker
from possible exosome release is virtually ruled out. Indeed, previous stud-
Europe) before addition to 106 BMDCs for 12 h.
ies have shown that productive infection by means of exosomes required
a minimum of five days of co-culturing of purified and concentrated exo- Prion infection of CGN cultures. Brain homogenate from 139A-infected mice
somes with recipient cells2. Nevertheless, to exclude exosome transfer in (at a final concentration of 0.01%) was added after sonication to the neuronal
our co-culture conditions, we set up co-cultures through translucent filters culture two days after plating and left for the whole experiment without washes,
as described previously36. PrPSc was also enriched from brain homogenate by
(pore size 0.4 µm) between loaded BMDCs and moRK13 cells (epithelial precipitation with sodium phosphotungstate (PTA) as described below before
RK13 cells expressing mouse PrP) that had previously been infected by exo- labelling with Alexa 546. CGN cultures were treated with either PTA or labelled
somes31. These filters physically separate BMDCs from moRK13 cells but PTA (Alexa-PrPSc), using amounts equivalent to 0.01% of 139A brain homogenate
allow the passage of exovesicles32. Using the same co-culturing conditions as under identical culturing conditions36.
for the neurons, we observed infection of moRK13 cells when co-cultured
Proteinase K assay. CGN or BMDC lysates were precleared by centrifugation,
with loaded BMDCs, in a similar manner to the neuronal cultures, but not treated with proteinase K (PK) and quenched with phenylmethylsulphonyl fluo-
when the filters were used (Supplementary Information, Fig. S7c). Similarly, ride. After precipitation with methanol, the pellet was resuspended in sample
studies showing transmission of prion infection by cell–cell contact were buffer before SDS–PAGE and western blot analysis with Sha31 and secondary
very inefficient and required a co-incubation time of at least one week32,33. anti-mouse antibody coupled to horseradish peroxidase. Immunoreactivity was
detected by enhanced chemiluminescence (ECL; Amersham).
In our case, the co-incubations were reduced to several hours with a ratio
of 1:4 loaded DCs to neurons, which is very low compared with the 1:1 BMDCs-CGN co-cultures. After several washes in IMDM and PBS, BMDCs
ratio used to study cell–cell spreading of infection32,33. Thus, although we loaded with 139A brain homogenate were co-incubated with CGN cultures, two
cannot rule out the existence of other mechanisms for prion spreading in days after being plated, at a ratio of 1:4 overnight. BMDCs were removed, and
the neuronal cultures were thoroughly washed in DMEM before incubation in
vivo, our data indicate that transfer of PrPSc through TNTs is an extremely
neuronal culture medium. As a control, neuronal cultures were incubated with
efficient way of spreading infection between neuronal cells and between 0.01% of brain homogenate. At 14 and 21 days after co-incubation, neuronal
BMDCs and neurons. cultures were lysed and subjected to PK assay.
Overall, our data show that TNTs can mediate the intercellular transfer
LysoTracker red and membrane dye staining for live imaging. For all experi-
of PrPSc in vitro between neuronal cells and between DCs and neurons.
ments, 150,000 CAD cells plated overnight were incubated either with LysoTracker
We therefore propose that TNTs could function in intercellular transfer of (Molecular Probes) (1:1,000 dilution) for 30 min at 37 °C or with 5-carboxyte-
PrPSc in vivo and participate in spreading infectious prions between neu- tramethylrhodamine succinimidyl ester (TAMRA; Invitrogen) (1:1,000 dilution)
rons within the brain and from the periphery to the CNS by establishing for 1 h at 37 °C or with 1,1´-dioctadecyl-3,3,3´,3´-tetramethylindocarbocyanine
neuroimmune interactions in lymphoid organs. Interestingly, CD14+ cells perchlorate (DiI; Invitrogen) (1:100 dilution) for 10 min at 37 °C, washed exten-
sively, and imaged at 40% confluence for better visualization of TNT.
(which contain a subpopulation of DCs) harbour most of the infectivity
in blood from orally and naturally infected sheep (Olivier Andreoletti, Immunofluorescence labelling. CAD cells plated overnight were washed care-
personal communication), possibly extending to blood the involvement fully, fixed with 2% paraformaldehyde (PFA) for 30 min at room temperature
of DCs in the spread of infection. Finally, the observation that patho- (20–22oC), quenched with NH4Cl and permeabilized with 0.1% Triton X-100 for
gens as diverse as viruses12,16, bacteria34 and now prions can exploit TNTs 2 min at room temperature. For fixed ScCAD cells, the cells were pretreated with
guanidinium (Gnd) as published23. Cells were then blocked with 2% BSA in PBS
and TNT-like structures for invading eukaryotic cells implies an ancient for 30 min at room temperature, and incubated with primary antibody (Sha31).
evolutionary origin for these structures and a function in intercellular The cells were carefully washed, incubated with AlexaFluor 488-conjugated sec-
communication that has hitherto been underestimated. ondary antibody and washed thoroughly before imaging.

letters
Co-culture conditions for transfer of endogenous PrPSc. A total of 75,000 CAD image processing received from the Plate-Forme Imagerie Dynamique at the
cells transiently transfected with Cherry–PLAP were co-cultured overnight with Pasteur Institut. K.G. is supported by the Pasteur Foundation Fellowship Program,
75,000 ScCADs. After 2 h, some of the dishes were incubated with 100 nM latrun- E.S. received a fellowship (2004-07) from the Bavarian Research Foundation (BFS),
culin A (Invitrogen) for the rest of the time. All the cells were then fixed with 2% D.B. received funding from the Fondation Canadienne Louis Pasteur, and Z.M.
received funding from Ile-de-France. This work was supported by grants to C.Z.
PFA, denatured with 6 M Gnd for 10 min and immunostained with Sha31 and
from the European Union (Strainbarrier (FP6 Contract No 023183 (Food)) and
with AlexaFluor 488 antibody for immunofluorescence analysis. from Telethon GGP0414.
Detection and quantification of endogenous PrPSc transfer. After combining Author contributions
a wavelet filtering technique with a statistical de-noising procedure, we fixed a C.Z. and E.S. conceived the project. K.G. and E.S. planned and performed most of
threshold on intensities above which the intensity was considered as originating the experiments with TNTs in different cells and analysed the data. C.L. planned
from a PrPSc signal. To compute this threshold automatically, the outline of a and performed the infection experiments and analysed the data. Z.M. and A.C.
reference infected cell (green outline) was first drawn for each field analysed. The planned and performed experiments in fixed CAD cells and analysed the data.
histogram of intensities was then automatically divided into two classes by the D.T.B. prepared the Alexa-PrPSc and discussed the experiments. N.C. and F.C.
k-means clustering technique. We fixed the threshold on intensities to identify performed most of the quantitative image analysis under the supervision of J.C.O.
J.E. helped with image reconstruction and discussed the data. A.M. prepared the
the presence of PrPSc aggregates as being the lowest value of the brightest class.
BMDCs and discussed the related experiments. D.M. co-directed the PhD thesis of
This threshold was applied to the de-noised intensities observed in the outline of E.S. and discussed data with E.S. and C.Z. C.Z. coordinated the project and assisted
the cytoplasm of the recipient CAD cell (red outline). If a value in the cytoplasm with planning the experiments and data analysis. K.G., E.S. and C.Z. wrote the
of the recipient cell was above this threshold, a PrPSc signal (green cross) was manuscript. All authors discussed the results and manuscript text.
detected and the cell was taken as positive for transfer of PrPSc.
Competing financial interests
Fluorescent labelling of PTA-precipitated PrPSc with AlexaFluor 546. Brain The authors declare that they have no competing financial interests.
homogenate was mixed 1:1 with 4% sarkosyl (Fluka)/PBS and agitated at 37 °C for
10 min. After treatment with benzonase (Sigma), the suspension was precleared Published online at http://www.nature.com/naturecellbiology
Reprints and permissions information is available online at http://npg.nature.com/
by centrifugation. After precipitation with PTA, pellets were pooled, resuspended
reprintsandpermissions/
in 0.1% sarkosyl/PBS to 1 mg ml−1, and AlexaFluor 546 carboxylic acid, succin-
imidyl ester (Invitrogen) solution (10 mg ml−1) was added to the suspension to 1. Mabbott, N. A. & MacPherson, G. G. Prions and their lethal journey to the brain. Nature
a final dye concentration of 1 mg ml−1 and agitated for 1 h at room temperature. Rev. Microbiol. 4, 201–11 (2006).
2. Fevrier, B. et al. Cells release prions in association with exosomes. Proc. Natl Acad.
The reaction was quenched with hydroxylamine and centrifuged, and the pellet Sci. USA 101, 9683–9688 (2004).
was washed. The fluorescently labelled pellet was resuspended with 0.1% sarko- 3. Leblanc, P. et al. Retrovirus infection strongly enhances scrapie infectivity release in
syl/PBS to a final concentration of 1 mg ml−1. For analysis, 1 µg of material was cell culture. EMBO J. 25, 2674–2685 (2006).
subjected to SDS–PAGE and imaged on a Typhoon Imager (GE Healthcare). Gels 4. Liu, T. et al. Intercellular transfer of the cellular prion protein. J. Biol. Chem. 277,
47671–47678 (2002).
were subsequently transferred to nitrocellulose by western blotting and revealed 5. Aucouturier, P. et al. Infected splenic dendritic cells are sufficient for prion transmission
with anti-PrP antibody (Sha31) and ECL (Amersham). to the CNS in mouse scrapie. J. Clin. Invest. 108, 703–708 (2001).
6. Montrasio, F. et al. Impaired prion replication in spleens of mice lacking functional
Loading of cells with Alexa-PrPSc. Alexa-PrPSc (1 µl) sonicated in 1 ml of BMDC follicular dendritic cells. Science 288, 1257–1259 (2000).
7. Prinz, M. et al. Lymph nodal prion replication and neuroinvasion in mice devoid of
medium was added to one well of the BMDCs in hydrophobic six-well plates (see
follicular dendritic cells. Proc. Natl Acad. Sci. USA 99, 919–924 (2002).
Supplementary Methods, ‘Bone-marrow derived dendritic cells’) overnight. CAD 8. Rustom, A., Saffrich, R., Markovic, I., Walther, P. & Gerdes, H. H. Nanotubular highways
cells were loaded overnight with 5 µl of sonicated Alexa-PrPSc, 24 h after plating. for intercellular organelle transport. Science 303, 1007–1010 (2004).
CAD cells were thoroughly washed before imaging or fixation. BMDCs were 9. Onfelt, B., Nedvetzki, S., Yanagi, K. & Davis, D. M. Cutting edge: Membrane nanotubes
connect immune cells. J. Immunol. 173, 1511–1513 (2004).
physically detached and resuspended in 30 ml of IMDM. The cells were washed
10. Hsiung, F., Ramirez-Weber, F. A., Iwaki, D. D. & Kornberg, T. B. Dependence of
thoroughly, resuspended in neuronal medium and added to the neuronal cultures Drosophila wing imaginal disc cytonemes on Decapentaplegic. Nature 437, 560–563
overnight before cell imaging. (2005).
11. Ramirez-Weber, F. A. & Kornberg, T. B. Cytonemes: cellular processes that project to the
Transfer of Alexa-PrPSc through TNTs. CAD cells loaded overnight with Alexa- principal signaling center in Drosophila imaginal discs. Cell 97, 599–607 (1999).
12. Sherer, N. M. et al. Retroviruses can establish filopodial bridges for efficient cell-to-cell
PrPSc were washed extensively and co-cultured with control CAD cells for 12 h. The transmission. Nature Cell Biol. 9, 310–315 (2007).
cells were then either imaged live or fixed with 2% PFA, denatured with 6 M Gnd and 13. Chinnery, H. R., Pearlman, E. & McMenamin, P. G. Cutting edge: Membrane nanotubes
immunostained with Sha31 and AlexaFluor 488-conjugated secondary antibody. in vivo: a feature of MHCII+ cells in the mouse cornea. J. Immunol. 180, 5779–5783
(2008).
Imaging systems. Fluorescence images were acquired with a microscope con- 14. Demontis, F. & Dahmann, C. Apical and lateral cell protrusions interconnect epithelial
cells in live Drosophila wing imaginal discs. Dev. Dyn. 236, 3408–3418 (2007).
focal Revolution Nipkow Spinning-Disk imaging system (Andor Technology). 15. Gerdes, H.-H., Bukoreshtliev, N. V. & Barroso, J. F. V. Tunneling nanotubes: a new route
Differential interference contrast pictures were acquired with Marianas imag- for the exchange of components between animal cells. FEBS Lett. 581, 2194–2201
ing system (TripleI) and Supplementary Movie S13 was acquired by confocal (2007).
microscopy on a Zeiss LSM 510. Long time-lapse movies were acquired with the 16. Sowinski, S. et al. Membrane nanotubes physically connect T cells over long dis-
tances presenting a novel route for HIV-1 transmission. Nature Cell Biol. 10, 212–219
Biostation IM from Nikon. Image analyses of the raw data were obtained with (2008).
ImageJ (http://rsb.info.nih.gov/ij/) and Osirix (Osirix Medical Imaging Software) 17. Qi, Y., Wang, J. K. T., McMillian, M. & Chikaraishi, D. M. Characterization of a CNS cell
software programs. line, CAD, in which morphological differentiation is initiated by serum deprivation. J.
Neurosci. 17, 1217–1225 (1997).
Tracking of vesicles and measurement of mean square displacement, diameter 18. Mahal, S. P. et al. Prion strain discrimination in cell culture: the cell panel assay. Proc.
Natl Acad. Sci. USA 104, 20908–20913 (2007).
and length. All these measurements were obtained with QUIA (QUantitative 19. Sherer, N. M. & Mothes, W. Cytonemes and tunneling nanotubules in cell–cell com-
Image Analysis) Software (http://www.bioimageanalysis.org). munication and viral pathogenesis. Trends Cell Biol. 18, 414–420 (2008).
20. Reed, B. C. et al. GLUT1CBP(TIP2/GIPC1) interactions with GLUT1 and myosin VI: evi-
Note: Supplementary Information is available on the Nature Cell Biology website. dence supporting an adapter function for GLUT1CBP. Mol. Biol. Cell 16, 4183–4201
(2005).
Acknowledgements 21. Howard, J. Molecular motors: structural adaptations to cellular functions. Nature 389,
We thank P. Lazarow, G. Guizzunti and C. Bowler for critical reading of the 561–567 (1997).
22. Magalhaes, A. C. et al. Uptake and neuritic transport of scrapie prion protein coincident
manuscript. We thank S. Blanchard and D. Bohl-Delfaud for their help in preparing
with infection of neuronal cells. J. Neurosci. 25, 5207–5216 (2005).
the GFP–PrPwt-retroviral vector, and P. Casanova and J. Vinatier for technical 23. Taraboulos, A., Serban, D. & Prusiner, S. B. Scrapie prion proteins accumulate in
help. We thank H. Laude, A. F. Hill, P. Cossart and M. Way for their gifts (cells, the cytoplasm of persistently infected cultured cells. J. Cell Biol. 110, 2117–2132
constructs and reagents). We are grateful for assistance with microscopes and (1990).

letters
24. Watkins, S. C. & Salter, R. D. Functional connectivity between immune cells mediated 32. Kanu, N. et al. Transfer of scrapie prion infectivity by cell contact in culture. Curr. Biol.
by tunneling nanotubules. Immunity 23, 309–318 (2005). 12, 523–530 (2002).
25. Huang, F. P., Farquhar, C. F., Mabbott, N. A., Bruce, M. E. & MacPherson, G. G. 33. Paquet, S. et al. Efficient dissemination of prions through preferential transmission to
Migrating intestinal dendritic cells transport PrPSc from the gut. J. Gen. Virol. 83, nearby cells. J. Gen. Virol. 88, 706–713 (2007).
267–271 (2002). 34. Onfelt, B. et al. Structurally distinct membrane nanotubes between human macro-
26. McBride, P. A. & Beekes, M. Pathological PrP is abundant in sympathetic and sensory phages support long-distance vesicular traffic or surfing of bacteria. J. Immunol. 177,
ganglia of hamsters fed with scrapie. Neurosci. Lett. 265, 135–138 (1999).
8476–8483 (2006).
27. Lambrecht, B. N. Immunologists getting nervous: neuropeptides, dendritic cells and
35. Brejot, T. et al. Forced expression of the motor neuron determinant HB9 in neural stem
T cell activation. Respir. Res. 2, 133–138 (2001).
cells affects neurogenesis. Exp. Neurol. 198, 167–182 (2006).
28. Defaweux, V. et al. Interfaces between dendritic cells, other immune cells, and nerve
fibres in mouse Peyer’s patches: potential sites for neuroinvasion in prion diseases. 36. Cronier, S., Laude, H. & Peyrin, J. M. Prions can infect primary cultured neurons
Microsc. Res. Tech. 66, 1–9 (2005). and astrocytes and promote neuronal cell death. Proc. Natl Acad. Sci. USA 101,
29. Dorban, G. et al. Oral scrapie infection modifies the homeostasis of Peyer’s patches’ 12271–12276 (2004).
dendritic cells. Histochem. Cell Biol. 128, 243–251 (2007). 37. Mederle, I. et al. Plasmidic versus insertional cloning of heterologous genes in
30. Luhr, K. et al. Scrapie protein degradation by cysteine proteases in CD11c+ dendritic Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune
cells and GT1-1 neuronal cells. J. Virol. 78, 4776–4782 (2004). responses. Infect. Immun. 70, 303–314 (2002).
31. Vella, L. J. et al. Packaging of prions into exosomes is associated with a novel pathway 38. Kaech, S. & Banker, G. Culturing hippocampal neurons. Nature Protocols 1, 2406–
of PrP processing. J. Pathol. 211, 582–590 (2007). 2415 (2006).

s u p p l e m e n ta r y i n f o r m at i o n
DOI: 10.1038/ncb1841
Figure S1 (a and b) Mean square displacement (MSD) measurements the vesicle enters the cell (b) The orange and green lines corresponding
suggest the involvement of molecular motors in vesicular transfer by to the fitted log MSDs curve for the TNT and inside the cell, respectively,
TNTs. (a) MSD data points of the LysoTracker vesicle in the TNT (blue approximate well the measured data points (blue and green triangles).
triangles) and in the cell (green triangles) are well fitted by a power law The slopes of the log MSD (∆t) curves are greater than 1 for vesicles in
relationship with respect to the time intervals ∆t as shown by the good the TNT (a = 1.704) and in the cell (a = 1.403), indicating again that the
linear approximation when working in logarithmic scales (orange and green vesicle has a directed movement throughout the movie, even inside the
solid lines). The slopes of these two lines are above 1, in the TNT (orange, a cell. (c) TNT contains GFP-PrPwt within vesicles of Lysosomal origin in
= 1.823) and in the cell (green, a = 1.544), demonstrating that the vesicle CAD cells. GFP-PrPwt transfected CAD-cells were labelled with LysoTracker.
has a directed movement throughout the movie. The shift in the Y-axis LysoTracker-positive vesicles, which in some cases also contain GFP-PrPwt
between the orange and green lines shows a slower directed movement after (arrow) were observed within TNTs. Scale bar represents 10 µm.
www.nature.com/naturecellbiology 1
Figure S2 Analysis of the Alexa-PrPSc preparations. The low molecular weight panel shows the same gel transferred to nitrocellulose by western blotting
contaminants in the Alexa 546-labeled PTA precipitate preparations do not and revealed with the PrP-specific Sha 31 antibody. (b) Fluorogram depicting
contain detectable protein. (a) Labelling of PrPSc with Alexa 546. PrPSc was bands excised from gel with samples as follows: A) Unlabelled PTA precipitate
isolated from 10% brain homogenates of strain 139A-infected mice by a that was not digested with proteinase K. B) Alexa 546-labelled PTA precipitate
modified protocol of phosphotungstic acid precipitation. The precipitates were that was not digested with proteinase K. C) unlabelled PTA precipitate
labelled with Alexa 546 carboxylic acid succinimidyl ester and resuspended digested with proteinase K. D) Proteinase K-digested PTA precipitate labelled
in 0.1% sarkosyl/PBS as detailed in Supplementary Methods. The left panel with Alexa 546. MW = molecular weight marker (kDa); BPB = bromophenol
is a fluorogram of 1 µg of the fluorescent preparation subjected to SDS-PAGE blue in the marker. (c) Table: Proteins identified in the indicated low molecular
and scanned on a Typhoon Imager at excitation/emission 532/580. The right weight bands (boxes in b) by MALDI-TOF M/S and MS/MS.
2 www.nature.com/naturecellbiology
Figure S3 Detection and quantitation of endogenous scrapie transfer. (green crosses) that were detected automatically. (b) One example of four
Transfer of endogenous PrPSc between ScCAD cells and Cherry-PLAP CAD different cases analysed is shown: (1) Cherry-PLAP transfected CAD cell
cells was analysed. (a) Diameters of TNTs in cells after fixation and Gnd (red outline) that do not touch the donor ScCAD cell (green outline). No
treatment. Twenty-three TNTs were analysed from three independent PrPSc is observed in the recipient cell. (2) Cherry-PLAP transfected CAD cell
experiments in co-cultures of Cherry-PLAP CAD cells and ScCAD cells, (red outline) in direct cell contact with the ScCAD cell (green outline). No
fixed, denatured and labelled with Sha31. The percent of cells as a function PrPSc is observed in the recipient cell. (3) Cherry-PLAP transfected CAD cell
of the average diameters of TNTs in nanometers are plotted. The TNTs that (red outline) in contact with the donor ScCAD cell via TNTs (green outline).
survive fixation and Gnd treatment are those with larger diameters (compare PrPSc signal (green cross) is observed in the recipient cell. (4) co-cultures
to Fig. 1d). All of the images were acquired with an Andor spinning disk treated with Latrunculin to block TNT formation. No PrPSc is observed in
confocal microscope (b-c) Fields of cells were taken in both green/red the recipient cell (red outline). (c) Three additional examples of endogenous
channels to discriminate between the infected and non-infected cells. PrPSc transfer via TNTs. PrPSc (green crosses) are found both in the
The green channel was selected for each Z-stack and the cytoplasm of the cytoplasm of recipient cells (red outline) and in the TNTs connecting them
recipient and donor cells were outlined in red or green respectively. “Fire” to the ScCAD cells. Two Z-stacks were chosen from the original 3D-stacks
Look-Up Table (LUT) images were chosen to better visualise PrPSc signals for all 3 examples. Scale bars represent 10 µm.
Figure S4 Immunohistofluorescence of murine spleen. Antibodies, spleen tissue from C57BL/6 mice. Arrows mark DCs in close proximity to
recognizing specific markers of DCs (anti-CD11c, red) and sympathetic neurites extending away from central arteriole (asterisk). Images were obtained
neurons (anti-Tyrosine-Hydroxylase, green) were utilised on cryo-cuts of by confocal microscopy on a Zeiss LSM 510. The scale bar represents 5 µm.
Figure S5 BMDCs interact via TNTs with differentiated CAD and with primary CGNs. The left panels are representative Z-stacks in the bottom of the 3D
CGNs. (a) Various examples of TNTs (black arrows) are shown between stack, the middle panels are representative Z-stacks in the middle of the 3D
BMDCs and differentiated CAD cells. (b) In co-cultures of BMDCs and stack, and the right panels are representative Z-stacks at the top of the 3D
primary CGNs, similar TNT connections (black arrows) are observed. The top stack. Images were acquired with a Marianas bright-field microscope (TripleI,
and lower panels are two examples of TNTs connecting BMDCs to primary Germany). The scale bars represent 10 µm.
Figure S6 Intercellular transfer of Alexa-PrPSc between Bone-marrow dendritic left) from Fig. 5c depict Alexa-PrPSc particles (yellow rectangles) inside a TNT
cells (BMDCs) and neurons via TNTs. BMDCs loaded with Alexa-PrPSc (red) are connecting the two cells (left lane), inside of the neurite (center) and at the
connected to hippocampal neurons, transduced with GFP-PrPwt-virus (green), distal end of another TNT (right lane). This suggests the intercellular transfer
via TNTs (white arrowheads). Different confocal layers (specified in the upper of Alexa-PrPSc from DCs to neurons via TNTs. Scale bar represents 5 µm.
Figure S7 BMDCs loaded with 139A brain homogenate pass infection to separates the two cell types. Lysates were PK digested before PrP-res
overnight co-cultured cells. (a) Efficient uptake of 139A brain homogenate could be revealed using Sha31. Control DC input from BMDCs loaded
by BMDCs. BMDCs incubated in the presence of 139A brain homogenate cells (Input loaded DC). The BMDCs loaded with 139A brain homogenate
(BMDC+139A) or without (BMDC alone) were analyzed for PrP-res content were either co-cultured with moRK13 cells directly (coculture) or plated
after PK digestion. Immunoblots revealed with Sha31 show the amount on a filter (filter). After an overnight co-culture, the BMDCs were removed
of PrP-res contained in 50 µg of infected brain tissue (139 alone) as a and after extensive washes with media, moRK13 cells were cultured for
control, compared to BMDCs loaded with 139A (efficient incorporation) or an additional 24 hours to 5 weeks. While moRK13 cells co-cultured with
non-loaded BMDCs (no PrP-res signal). (b) PrPSc degradation in BMDCs. BMDCs show a PrP-res signal after 24 hours, no signal was visible in
BMDCs were loaded with 139A brain homogenate (I) or normal brain the moRK13 cells when the co-cultures were separated by a filter (see
homogenate (NI). Following incubation for the indicated times, BMDC Supplementary Methods). Similarly, PrP-res signal accumulation was
lysates were PK digested before PrP-res immunoblot analysis with Sha31. observed after 5 passages in the direct co-cultures, while no signal was
PrP-res acquired by BMDCs is degraded after 72 hours of culture, as shown visible when a filter was used, suggesting that infection of moRK13 cells
by the decreased PrP-res signal at this time point. (c) Loaded BMDCs was not due to exosomal release. Control of moRK13 cell extracts from
infect moRK13 cells in direct co-cultures but not when a filter physically non-infected cells (moRK13 NI) shows no PrP-res signal.
Figure S8 Full-length blots from Figures 3c and 5d. (a-c) Cerebellar cerebellar granule neurons (CGN) in co-cultures with BMDCs loaded with
granule neurons were exposed to brain homogenate (139A) (a), brain 139A brain homogenate. PrP-res analysis after PK digestion was performed
homogenate purified by sodium phosphotungstic acid (PTA) (b) or Alexa- on CGN cultures after 14 or 21 days post-co-culture with BMDCs loaded
labelled PTA (Alexa-PrPSc) (c). Duplicate experiments were loaded for each with brain homogenate (DC co-culture) or after direct exposure to brain
condition. Cell lysates from CGN cultures after 7, 14 and 21 days were homogenate (139A). In both cases, PrP-res accumulation is observed in
PK-treated and PrP-res was detected by immunoblotting using Sha31. CGN cultures after 21 days. No signal could be detected in non-infected
PrP-res accumulation is observed overtime for all conditions. The lanes cells (NI). The lanes with a yellow star are the cropped lanes shown in Fig.
with a yellow star are the cropped lanes shown in Fig. 3-c. (d) Infection of 5-d. MW = molecular weight marker (kDa).
Supplementary Movie Legends
Movie S1 Traffic of LysoTracker vesicles via TNTs in CAD co-cultures. CAD cells were treated as described in Fig. 2a. Movies of the CAD cells co-cultures
were obtained using an Andor confocal spinning disk microscope. The cells were incubated in the microscope chamber with temperature control, and
imaged overtime, every 25 sec for 23 minutes. For each time points, multiple Z-stacks were acquired. Projections were obtained for each time points
using the ImageJ software. The movie represents two concatenated movies. Overtime, a LysoTracker positive vesicle can be seen moving toward a GFP-actin
transfected cell (green) in a directed movement, and entering its cytoplasm, where the vesicle continues to move in a directed movement (see Fig.2). The
diameter of this TNT along its length averaged 457 nm (+/- 107).
Movie S2 Tracking of a LysoTracker vesicle within TNTs and exiting in a recipient cell. Two-dimensional tracking of Movie S1 (see Supplementary Methods)
allows for the determination of the speed and improves visualization of the vesicle’s movements both inside the TNT and after entering the cell.
Movie S3 Projection movie of CAD cells connected by a network of TNTs overtime. CAD cells were treated as described in Fig. 2d. Multiple Z-stacks were
obtained for each time points. Maximum projections for each frame were obtained with the ImageJ software. Movie S3 is 20 minutes long with 10 sec
between each frame. Overtime, the movement of the cells, and the TNTs is clearly visible, along with the occasional collapse of some TNTs. However, most
TNTs were able to retain their connections to the cells over the entire length of the movie. As can be seen in the cell on the right lower corner, a vesicle is
travelling through the TNT and entering the cell.
Movie S4 Movie of GFP-PrP vesicle movement within a TNT. The frames from Movie S3, corresponding to the lower right cell, were selected. Similar to Movie
S3, the movie represent time points every 10 sec for 220 sec. The vesicle observed in Movie S3, can be visualised, going down the TNT and entering the cell.
The diameter of this TNT along its length averaged 338 nm (+/- 103).
Movie S5 Tracking of GFP-PrP vesicle movement within a TNT. Three-dimensional tracking of Movie S4 (see Supplementary Methods) allows for a
determination of the speed and improves visualization of the vesicle’s movements inside the TNT and as it enters the cell.
Movie S6 Movement of Alexa-PrPSc vesicles toward and inside a TNT. CAD cells loaded with Alexa-PrPSc (see Supplementary methods) were imaged in a live
microscope chamber using an Andor confocal spinning disk microscope. Z-stacks of 0.25 µm steps were acquired for each time points for 30 minutes every
15 seconds. Movement a numerous small vesicles can be seen entering the TNT and trafficking down the tube.
Movie S7 Zoomed-in view of Movie S6 to better follow the movement of Alexa-PrPSc vesicles. The upper part of Movie S6 (loaded cell with top part of TNT)
have been cropped and re-sized to better visualise the movement of multiple Alexa-PrPSc vesicles going from the top cell inside and through the top part of
the TNT.
Movie S8 TNT connection between BMDCs and dendrites of differentiated CAD cells. CAD cells were differentiated for one week by serum starvation. The
cells were plated onto Ibidi dishes and co-cultured with BMDCs. Twenty-four hours post-co-culturing, BMDCs were imaged by wide-field microscopy (see
Supplementary Methods). A single Z stack was acquired every minute for 38 minutes. A TNT is observed between a BMDC and the neurite of a differentiated
CAD cell. Overtime, the tube appears to move along the side of the dendrite, while maintaining the two cell types in contact with one another.
Movie S9 TNT connection between BMDCs and differentiated CAD cells. CAD cells were differentiated for one week by serum starvation. The cells
were plated onto Ibidi dishes and co-cultured with BMDCs. Twenty-four hours post-co-culturing, BMDCs were imaged by wide-field microscopy (see
Supplementary Methods). A single Z stack was acquired every 30 seconds for 28.5 minutes. A TNT is observed between a BMDC and the cell body of a
differentiated CAD cell.
Movie S10 Time-lapse analyses of co-cultures of differentiated CAD cells with BMDCs reveal continuous TNT formations between cells. Serum deprived
differentiated CAD cells were co-cultured with BMDCs and imaged with the Time-lapse Imaging System Biostation IM from Nikon (see Supplementary
Methods). Immediately after adding the BMDCs to the differentiated CAD cells (plated overnight on an Ibidi dish), the co-cultured cells were incubated into
the Biostation chamber (Temperature, humidity and CO2 controlled). The cells were left for 30 minutes within the chamber to equilibrate. Multiple fields
were taken (single Z-stack) every 5 minutes for 24 hours. Movie S10 is a representative movie, edited in time. In this time frame (6 hours), CAD cells are
much more mobiles compare to BMDCs (label DCs in the movie). Multiples TNTs (asterisks) can be observed between cells of the same and different origins.
Scale bar represents 10 µm.
Movie S11 Time-lapse analyses of co-cultures of differentiated CAD cells with BMDCs reveal continuous TNT formations between cells. Serum deprived
differentiated CAD cells were co-cultured with BMDCs and imaged with the Time-lapse Imaging System Biostation IM from Nikon (see Supplementary
methods). Immediately after adding the BMDCs to the differentiated CAD cells (plated overnight on an Ibidi dish), the co-cultured cells were incubated into
the Biostation chamber (Temperature, humidity and CO2 controlled). The cells were left for 30 minutes within the chamber to equilibrate. Multiple fields
were taken (single Z-stack) every 5 minutes for 24 hours. Similar to Movie S10, this movie is a representative movie, edited in time. In this time frame (7.5
hours), CAD cells are much more mobiles compare to BMDCs (label DCs in the movie). Multiples TNTs (asterisks) can be observed between cells of the same
and different origins. Scale bar represents 10 µm.
Movie S12 DIC Z-stacks from bottom to top of a BMDC connected to primary CGNs via a network of TNTs. BMDCs were added to a culture of CGNs plated on
Ibidi dishes and co-cultured overnight. The dish was put on a 37oC, 5% CO2 microscope chamber and sequential DIC/fluorescence Z-stacks (0.35 µm steps)
were obtained from the bottom to the top, using a Marianas bright-field microscope (TripleI, Germany). TNTs connecting the BMDC and CGNs can be seen, as
we go through the Z-stack from bottom to top.
Movie S13 Connections between primary hippocampal neuron and bone marrow derived dendritic cell are not attached to the substratum. Co-culture of
neurons with dendritic cells were labelled with TAMRA (staining proteins on the membrane) and imaged by confocal microscopy. Shown is a whole Z-scan,
starting at the bottom of the substratum and ending with the top of the DC. Note that the fluorescence inside of the TNT moves with each confocal layer
(thickness 300 nm). This demonstrates that the connection is not attached to the substratum. The movie was done with LSM 510 software and Image J. The
scale bar represents 5 µm.
Supplementary Methods:
Primary cultures of cerebellar granule neurons (CGNs)
Primary cultures of cerebellar granule neurons (CGNs) were established as previously
described1. Briefly, CGNs were extracted from brains of 6 days old C57BL/6 mice by
enzymatic and mechanical dissociations. They were plated at a density of 800,000
cells/well on 12-well plates coated with 10 µg/mL of poly-D-lysine (Sigma) and
cultivated in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal calf
serum, 20mM KCl, penicillin, streptomycin (Gibco) and complemented with B27 and N2
supplement (Gibco). Cultures were incubated at 37°C in humidified atmosphere with 5%
CO2 and were complemented weekly with 1mg/mL Glucose and 10µM of the anti-
mitotics uridine and fluorodeoxyuridine (Sigma) to avoid astrocyte proliferation.
Bone-marrow derived dendritic cells (BMDCs)

BMDCs were differentiated from bone-marrow cells from 6- to 8-week-old C57 BL6
mice according to a method adapted from Méderlé et al.2. Briefly, bone-marrow cells
were seeded at 2x106 cells per 100 mm diameter Petri dish (Falcon, Becton Dickinson
Labware, Franklin Lakes, NJ) in 10 ml of Iscove's modified Dulbecco's medium (IMDM;
BioWhittaker Europe, Verviers, Belgium) supplemented with 10% heat-inactivated foetal
calf serum (FCS; Dutscher, Brumath, France), 1.5% supernatant from a J558 cell line
producing murine GM-CSF3, 50 U/ml penicillin, 50 µg/ml streptomycin, 50 µM 2-
mercaptoethanol and 2 mM glutamine (complete IMDM). Cultures were incubated at
37°C in a humidified atmosphere with 7% CO2. On day 3, 10 ml of complete IMDM was
added. On day 6, suspended cells and loosely adherent cells were harvested using
prewarmed 1% EDTA in Dulbecco's PBS without Ca2+ and Mg2+ (Biochrom AG,
Berlin, Germany). The recovered cells were further cultured under the same conditions as
above. On day 10, cells were harvested with EDTA as above and distributed in
hydrophobic 6-well plates (Evergreen Scientific, Los Angeles, CA) at a concentration of
9x105 cells/well in 3 ml complete IMDM.
Embryonic hippocampal neurons

Embryonic hippocampal neurons produced from C57/BL6 mice as previously described4
were grown in B27 supplemented medium on glass bottom culture dishes (MatTek, USA)
coated with poly-L-lysine (Sigma) according to the manufacturer’s instructions for 2
days. Neurons were transduced with virus encoding GFP-PrPwt and cultured for another
3 days. Subsequently, cells were utilised for co-culture experiments with BMDCs.
PK assay:
Following incubation for the indicated time with the different infectious prion
preparations, cells (BMDCs, CGNs or moRK13) were washed in PBS before lysis in TL1
buffer (Tris pH 7.4, 50mM; Sodium deoxycholate 0.5% and Triton X-100 0.5%). Neuron
and BMDC lysates were pre-cleared by centrifugation (1000 rpm, 5min.) and 50 µg of
proteins per cell lysates were treated with 2 µg or 0.5 µg of proteinase K (PK) for the
BMDC lysate or neuron lysate respectively, for 30 minutes at 37°C before stopping the
digestion with 5mM of PMSF. Next, the proteins were methanol precipitated for 1 hour at
-20°C before centrifugation at 12000 rpm for 30min. Pellet were resuspended in sample
buffer before analysis by SDS PAGE in 12% acrylamide gels and Western Blot with
Sha31 and secondary anti mouse antibody coupled to horseradish peroxydase.
moRK13 infections with BMDCs loaded with 139A brain homogenate

BMDCs were loaded with 5 mg of 139A brain homogenate overnight and then washed
three times in IMDM before culturing with moRK13 for 12 hrs. Loaded BMDCs were
directly added to moRK13 (co-culture) or plated on top of translucent 12mm diameter
Polyester filters (0.4 µm high pore density; from Costar- Corning) (filter), which allows
for the passage of exovesicles but blocks cell-to-cell contact. The input BMDCs (from
direct co-cultures and plated on the filters) were then removed from the moRK13 cultures
and lysed in TL1, digested with PK, analyzed by Western blot and revealed with Sha31
antibody. PK assays were also performed on moRK13 overtime to detect PrPres (either
24 hours or 5 weeks post-co-incubation) by similar Western blot analysis.
Fluorescent Labelling of PTA-precipitated PrPSc with Alexa-546

Brain homogenates were prepared by resuspending 139A-infected brains in PBS to 10%
(w/v) and performing 10 strokes of dounce homogenisation. This suspension was further

homogenised by passage through a 26 gauge needle 10 times, mixed 1:1 with 4%
sarkosyl (Fluka)/PBS (in 2 mL tubes) and agitated at 37 °C for 10 minutes. Next,
benzonase (Sigma) was added to a final concentration of 50U/mL and agitated at 37 °C
for 10 minutes. The suspension was pre-cleared by centrifugation at 18 000 x g for 10
minutes at room temperature (RT). The supernatant was removed and re-warmed to 37
°C. Next, a solution of 4% phosphotungstic acid (Sigma)/170 mM MgCl2 at 37°C was
added to this suspension to a final concentration of 0.3% and agitated for 10 minutes at
37 °C. This mixture was centrifuged for 10 minutes at 18 000 x g at RT. The supernatant
was removed and discarded. The pellets were washed twice with 2 mL of 0.1%
sarkosyl/PBS with centrifugation for 5 min at 18 000 x g. Pellets were pooled and
resuspended in 0.1% sarkosyl/PBS, sonicated in a cuphorn sonicator (Sonics) at 80%
amplitude with 5s pulses and 1s pauses for 1 minute and the protein concentration
measured by Micro BCA protein assay (Pierce); the suspensions were diluted to 1 mg/mL
with 0.1% sarkosyl/PBS. Alexafluor 546 carboxylic acid, succinimidyl ester (Invitrogen)
was resuspended to 10 mg/mL in DMSO and added to the suspension at 1:10 (vol/vol) to
a final dye concentration of 1mg/mL and agitated for 1 h at RT. The reaction was
quenched with 1 volume of 1.5M hydroxylamine (pH 8.5) overnight at 4 °C. The
suspension was centrifuged at 18 000 x g for 10 minutes at RT and the supernatant
removed and discarded. The pellet was washed 4 times with 1 mL 0.1% sarkosyl/PBS
with centrifugation steps of 18 000 x g for 10 minutes at RT. The fluorescently labeled
pellet was resuspended with 0.1% sarkosyl/PBS to a final concentration of 1 mg/mL and
sonicated for 30s at 80% amplitude with 5s pulses and 1s pauses. For analysis, one
microgram of material was subjected to SDS-PAGE and imaged on a Typhoon Imager
(GE Healthcare) at ex/em 532nm/580nm.
Mass Spectrometry of Low Molecular Weight Contaminant Bands of the Alexa-

PrPSc preparation, Co-Migrating With the Dye Front.
Five micrograms of material from each of the 4 following samples were subjected to
SDS-PAGE under clean conditions: A) PrPSc precipitated from brain homogenates of
139A-infected C57BL/6 mice by PTA that was not digested with proteinase K (PK) nor
labeled with Alexa 546; B) PTA precipitates not digested with PK but labeled with Alexa

546 (the preparation in use); C) PK-digested, unlabeled PTA precipitate; and finally D)
PK-digested PTA precipitate labeled with Alexa 546.
The gel was imaged without removal of the glass gel plates on a Typhoon Imager at
ex/em 532nm/580nm for visualisation of the fluorescent samples and at ex/em
633nm/670nm for visualisation of the molecular weight marker. Next, the lower
molecular weight bands were excised and submitted for mass spectrometry analysis by
MALDI-TOF MS and MS/MS. Peptide fingerprints were identified using the MASCOT
database. We obtained data with both Alexa-PrPSc (PK-) and Alexa-PrPSc (PK+). No
differences were observed within cells but the contaminant bands were bigger for the
PK+ samples. For this reason, in this manuscript, we only showed data pertaining to
experiments using PK- preparations. PrPSc content of PK- preparation was verified by
epitope retrieval using guanidium chloride followed by immunofluorescence using anti-
prion antibody Sha31 (Supplementary Fig. 2).
Loading of cells with Alexa-PrPSc

In all microscopy experiments utilizing Alexa-PrPSc, CAD cells were physically detached
(not trypsinised) and 150,000 CAD cells were seeded on Ibidi dishes overnight. One
microliter of our Alexa-PrPSc was sonicated in 1 ml of CAD media (80% amplitude for 2
minutes with pulses of 5 seconds, and pauses of 1 second) and incubated with the CAD
cells in the Ibidi dishes overnight. The cells were thoroughly washed prior to live cell
imaging or fixation, according to the experiments.
BMDCs destined to be used for microscopy were loaded with 1 µl of our Alexa-PrPSc
after sonication in 1 ml of BMDC media (80% amplitude for 2 minutes with pulses of 5
seconds, and pauses of 1 second) and added to one well of the BMDCs in hydrophobic 6-
well plates (see Supplementary Methods “Bone-marrow derived dendritic cells”, above)
overnight. The cells were collected by detaching them off the well with cell scrappers,
and resuspended in 30 ml of IMDM. The cells were spun down and resuspended in 30
ml of IMDM. After three 30 ml IMDM washes, the cells were resuspended in the
neuronal media and added to the neuronal culture overnight prior to cell imaging.
Transfer of infected brain homogenate via TNTs

CAD cells loaded for 12 hours with 1% brain homogenate from 139A infected mice were
extensively washed and split 3 days post-loading. Five days post-loading, the cells were
co-cultured overnight with CAD cells transiently transfected with GFP-GPI then fixed
with 2% PFA, denaturated with 6M Gnd and immunostained with Sha31.
Imaging systems
Live cell fluorescence images were acquired with a microscope confocal Andor
Revolution Nipkow-Spinning Disk imaging system (Belfast, Ireland). The Andor
technology was installed on a Zeiss axiovert 200M microscope, equipped with an Andor
EMCCD DV885 camera, three diode pumped solid state lasers with excitation at 405,
488 and 560 nm, a piezo mono-objectif for fast 3D acquisitions, and a confocal head
Spinning-disk Yokogawa CSU22. Images were acquired with an oil 63X Plan-
APOCHROMAT objective (1.4 NA) and with a 1X1 binning. All Z-stacks were
acquired at maximum speed of the microscope with Z-steps of 0.25 µm except Movie S1,
which had a Z-step of 0.30 µm. The time between each time points was determined based
on the amount of Z-stacks taken. In the representative movies described in this
manuscript, the time points were taken every 10 or 25 sec, as described in the figure
legends. With the Marianas bright-field system (TripleI, Germany) we used a 63X oil
objective (1.4 NA). These three imaging systems for living cells were equipped with a
heated control chamber kept at 37°C (Pecon, Germany). The fully automated Time-lapse
Imaging system Biostation IM from Nikon (Kanagawa, Japan) combines an incubator,
(temperature, humidity and CO2 control) and a built-in 2.0 megapixel monochrome CCD
camera. DIC images were acquired every 5 minutes with a 40X lens magnification.
Tracking of vesicles
In the acquired 3D confocal images the vesicles are inside TNTs, therefore the measured
intensities are the mixture of photons originated from vesicles and TNTs, which
disqualifies the use of a standard intensity-based vesicle detection technique. Our
approach5 to vesicle detection and tracking is adapted to this case since we are able to
separate intensities coming from vesicles and TNTs, before detecting and tracking
vesicles. The detection step exploits the different morphologies of the TNTs and vesicles
signals to build two separated images: images from isotropic signals that correspond to

vesicle intensities, and images from elongated signals that contain the intensities
produced by the TNTs. Once the images from the vesicles have been recovered, we can
detect accurately the 3D positions of the vesicles based on the intensities within the
TNTs. Finally, the tracking algorithm links detections from different time by modelling
the motion of the vesicles with an adaptive mixture of Brownian and directed models of
movement.
The cells border was automatically delimited by a 3D active mesh extraction procedure6.
By combining this surface representation with tracks of vesicles we can visualise and
study the entry of a vesicle in a cell cytoplasm.
Based on the extracted locations of vesicles across time, we also calculated the mean
speed and MSD curves. For detailed description of MSD please refer to Figure Legend
S2 and MSD Supplementary information. Tracking measurements were computed using
QUIA (QUantitative Image Analysis) Software (http://www.bioimageanalysis.org).
Length measurements of TNTs

Length measurements were computed using QUIA (QUantitative Image Analysis)
Software. The coordinates of the points of a tunnel were extracted in order to determine
its length. This was done in a semi-automatic way described as follows:
Denoising: we applied a denoising algorithm7, which is adapted to eliminate a mixture of
photon and readout noises. At the end of this step, the noise is well separated from the
image and the signal has better contrast. Extraction of the points, which belongs to the
tube: the user manually draws over the 2D tube an ROI on a xy view. This ROI is then
projected on the tube in z according to the intensity-weighted centroids.
Diameter measurements of TNTs

Diameter measurements were computed using QUIA (QUantitative Image Analysis)
Software. The mean tunnel diameter was measured as the average of the diameters at 5-
10 different positions along the tunnel. For each chosen position, the corresponding YZ
slice information was extracted. We model the tunnel as a 3D Gate line with a width
parameter sigma0 unknown. Our goal is to estimate this sigma0 and the tunnel diameter
is then estimated as 2*sigma0. Toward this goal, we used a Gaussian PSF approximation

model for the diffraction limit of the microscope8. The parameters for the Gaussian PSF
approximation are computed using the method described previously8, which use the same
configuration for the acquisitions. For the current model, the microscope aberrations have
not been considered.
The gate is convolved by the PSF represented as a Gaussian function. The residual is the
squared-difference between the input signal and the convolved one. The residual is
computed depending on sigma and the amplitude of the gate. For the smallest residual,
we keep sigma.
Since the image contains unwanted photon and readout noises, the fit was actually done
in a variance-stabilising transform domain. This transform is proposed in reference 10 to
stabilise and gaussianise the noise. Due to the PSF8, any object of a size smaller than 186
nm cannot be differentiated: their residual will always be the same. In addition, by using
fluorescent pictures, we could only calculate the average upper limit of our TNT
diameters.
Mean square displacement (MSD) measurements of vesicular transfer in TNTs. The

definition of MSD is given by MSD( t) := <|x(t+ t) – x(t)|2>. Here, |x(t+ t) – x(t)| is the
distance travelled by the object over a time interval of duration t, and the squared
magnitude is averaged (as indicated by the angle brackets) over many such time intervals.
The MSD is usually fitted by a power law, i.e., f( t) := C ta. By using logarithmic
scales the relationship between log (f( t)) and log ( t) is shown to be linear: log (f( t))
= log(C)+ a log ( t). The exponent a typically characterises the movement. If an object is
undergoing a free Brownian motion, its MSD will exhibit a linear relation, (i.e., a = 1
with C proportional to the diffusion constant). A super-linear relation (a > 1) indicates
actively transported objects showing directional movements; a sub-linear growth (a < 1)
indicates confined diffusive movements. In order to calculate the parameters (C, a) we
computed a fitted log MSD-data set from the raw log MSD-data set (shown in A and B).
As shown by the linear dependency between log (f( t)) and log( t) the a exponents
corresponds to the slope of the presented curves and the C values correspond to the f( t)
values for which the curves intersect the vertical axis.

Detection and Quantitation of scrapie transfer from infected to non-infected cells.
In order to detect the presence of PrPSc aggregates in cells, individual Z stacks were
extracted from the 3D stack and a dedicated detection procedure was performed. First we
enhanced isotropic shapes of the scale of the typical size of an aggregate, flattened the
intensity of the cell body and corrected the defects of illumination. This is done in a
single pass by combining a wavelet filtering technique of the image with a statistical de-
noising procedure that keeps only significant values. Once the corrected and de-noised
image is built only intensities coming from PrPSc and isotropic like structures in the cells
remain.
In order to decide if transfer of PrPSc has occurred, we determined if in the cytoplasm of
the non-infected CAD cells some intensities are the result of the presence of at least one
PrPSc aggregate. Assuming that PrPSc aggregates emit the brightest light in the cell, we
fixed a threshold on intensities above which the intensity is considered as originating
from a PrPSc signal. To automatically compute this threshold, the outline of a reference-
infected cell (green outline), with clear PrPSc aggregate signal intensities in its cytoplasm,
is first drawn for each field analysed. The histogram of intensities is then automatically
divided in two classes by the k-means clustering technique, the brightest class
corresponding to PrPSc intensities. We therefore fix the threshold on intensities to decide
the presence of a PrPSc as the lowest value of the brightest class. This threshold is then
applied to the de-noised intensities observed in the outline of the cytoplasm of the
recipient CAD cell (red outline). If a value in the cytoplasm of the recipient cell is above
this threshold the presence of a PrPSc signal (green cross) is detected and the cell is taken
as positive for transfer of PrPSc. We automatically recomputed this threshold for each
field of view analysed since the condition of illumination and the efficiency of the
fluorophore can vary between experiments. Statistical analysis correlating the presence of
TNTs and the presence of PrPSc in naïve cells was performed using Fisher’s exact test. To
avoid the type I error associated with multiple comparisons we applied a Bonferroni
correction and considered statistical significance to occur at p < 1.67x10-2.
Using this method we had a strong correlation between the presence of TNTs after
fixation and the transfer of PrPSc aggregates in the recipient cells. However we did not
observe PrPSc aggregates in cells that had been in contact via TNTs during the overnight

cultures, as might have been expected due to the transient nature of TNTs9. This could
result from three major not mutually exclusive reasons. First we used a high fluorescence
threshold to detect only the larger aggregates bona-fide PrPSc signal. Second, in cells still
connected via TNTs large aggregates are easily recognizable, while after transfer PrPSc
aggregates might disaggregate into smaller size particles and no longer be detectable with
our high threshold. This has been previously shown to occur after the uptake of
fluorescently labelled PrP-res aggregates10. In addition, we observed that after fixation
and Gnd treatment the remaining TNTs are the ones with larger diameters
(Supplementary Fig. 3a compare to Fig. 1d). These data, along with our live experiments
where transport was observed only in TNTs with a diameter above 300 nm (Movie S1
and Movie S4) suggest that only larger TNTs might be able to transport large PrPSc
aggregates. This would be in agreement with the finding in macrophages where vesicular
transport was observed only in thick TNTs11.
References:
1. Cronier, S., Laude, H., & Peyrin, JM. Prions can infect primary cultured neurons
and astrocytes and promote neuronal cell death. Proc. Natl. Acad. Sci. USA. 101,
12271-6 (2004).
2. Mederle, I. et al. Plasmidic versus insertional cloning of heterologous genes in
Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune
responses. Infect Immun 70, 303-14 (2002).
3. Zal, T., Volkmann, A. & Stockinger, B. Mechanisms of tolerance induction in
major histocompatibility complex class II-restricted T cells specific for a blood-
borne self-antigen. J Exp Med 180, 2089-99 (1994).
4. Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat Protoc 1, 2406-15
(2006).
5. Chenouard N., Bloch I., Olivo-Marin J.-C. Morphological source separation for
particle tracking in complex biological environments. To be presented at the
IAPR International Conference on Pattern Recognition, Tampa, FL, 8-11
December, 2008.
6. Dufour, A. & Olivo-Marin, J.-C. Tracking live cells in 4D microscopy: active
surfaces vs. active meshes. Proceedings of the 42nd Asilomar Conference on
Signals, Systems & Computers (2008, in press).
7. Zhang, B., Zerubia, J. & Olivo-Marin, J. C. Gaussian approximations of
fluorescence microscope point-spread function models. Appl Opt 46, 1819-29
(2007).
8. De Moraes Marim, M., Zhang, B., Olivo-Marin, Olivo-Marin, J-C. & Zimmer, C.
Improving single particle localization with an empirically calibrated Gaussian
kernel. ISBI 2008: 1003-1006.

9. Gurke, S. et al. Tunneling nanotube (TNT)-like structures facilitate a constitutive,
actomyosin-dependent exchange of endocytic organelles between normal rat
kidney cells. Exp. Cell. Res. 314:3669-83 (2008).
10. Magalhaes, A. C. et al. Uptake and neuritic transport of scrapie prion protein
coincident with infection of neuronal cells. J Neurosci 25, 5207-16 (2005).
11. Onfelt, B. et al. Structurally distinct membrane nanotubes between human
macrophages support long-distance vesicular traffic or surfing of bacteria. J
Immunol 177, 8476-83 (2006).

Figura 3 PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Figura 3 PDF

Uploaded by

Copyright:

Available Formats

letters

Prions hijack tunnelling nanotubes for intercellular

328 nature cell biology volume 11 | number 3 | MARCH 2009

Percentage of CAD cells

0 0 Less than 248 459 827

Average length of TNTs (µm) Average diameter of TNTs (nm)

nature cell biology volume 11 | number 3 | MARCH 2009 329

Velocity (nm s–1)

Velocity (nm s–1)

120 s 140 s 160 s

180 s 200 s 220 s

330 nature cell biology volume 11 | number 3 | MARCH 2009

GFP–GPI / 139A PrPSc

Alexa-PrPSc / PrP/ + Gnd

Alexa-PrPSc / PrP GFP–GPI

c 139A PTA Alexa-PrPSc

nature cell biology volume 11 | number 3 | MARCH 2009 331

PrPSc Cherry–PLAP Merge

Percentage of CADs containing PrPSc

332 nature cell biology volume 11 | number 3 | MARCH 2009

Days: 14 14 21 14 21 Merge Merge

nature cell biology volume 11 | number 3 | MARCH 2009 333

(Fig. 5c, arrowheads) and we observed Alexa-PrPSc in the TNT itself, Methods

334 nature cell biology volume 11 | number 3 | MARCH 2009

nature cell biology volume 11 | number 3 | MARCH 2009 335

336 nature cell biology volume 11 | number 3 | MARCH 2009

Supplementary Movie Legends

Bone-marrow derived dendritic cells (BMDCs)

Embryonic hippocampal neurons

© 2009 Macmillan Publishers Limited. All rights reserved.

moRK13 infections with BMDCs loaded with 139A brain homogenate

Fluorescent Labelling of PTA-precipitated PrPSc with Alexa-546

© 2009 Macmillan Publishers Limited. All rights reserved.

Mass Spectrometry of Low Molecular Weight Contaminant Bands of the Alexa-

© 2009 Macmillan Publishers Limited. All rights reserved.

Loading of cells with Alexa-PrPSc

Transfer of infected brain homogenate via TNTs

© 2009 Macmillan Publishers Limited. All rights reserved.

© 2009 Macmillan Publishers Limited. All rights reserved.

Length measurements of TNTs

Diameter measurements of TNTs

© 2009 Macmillan Publishers Limited. All rights reserved.

Mean square displacement (MSD) measurements of vesicular transfer in TNTs. The

© 2009 Macmillan Publishers Limited. All rights reserved.

© 2009 Macmillan Publishers Limited. All rights reserved.

© 2009 Macmillan Publishers Limited. All rights reserved.

© 2009 Macmillan Publishers Limited. All rights reserved.

You might also like

328 nature cell biology volume 11 | number 3 | MARCH 2009

330 nature cell biology volume 11 | number 3 | MARCH 2009

332 nature cell biology volume 11 | number 3 | MARCH 2009

(Fig. 5c, arrowheads) and we observed Alexa-PrPSc in the TNT itself, Methods

334 nature cell biology volume 11 | number 3 | MARCH 2009

336 nature cell biology volume 11 | number 3 | MARCH 2009