Recent observations have revealed that intercellular connections can be formed through membrane nanotubes. These delicate structures could facilitate transport of organelles and membrane proteins between cells. The sharing of cell surface and cytoplasmic components between cells could be commonplace in biology, but an important physiological role for membrane nanotubes between immune cells is difficult to test with current technology.

Rustom et al. [1] have recently reported the existence of nanotubes formed by continuous membrane that could network various cultured cells and function as channels for organelle transport. Similarly, membrane nanotubes can also be observed to connect various immune cells, including Epstein–Barr virus-transformed B-cells, primary macrophages and natural killer cells [2]. For example, membrane proteins can be transferred between two 721.221 B-cells through naonotubes connected to those cells (Figure 1). Here, we also show that lipid organelles can be visualized inside nanotubes formed between HEK-293T (human embryonic kidney-293T) cells (Figure 2). Membrane tethers can also be seen in earlier micrographs depicting the disassembly of cytotoxic T lymphocytes or natural killer cell immunological synapses [3,4]. Membrane bridges creating channels with a diameter of 50–100 nm between cytotoxic T lymphocytes and target cells [4] are possibly a precursor structure for larger membrane nanotubes. Cell-membrane tethers have also been induced mechanically [5] or by applying a flow [6].

Membrane proteins can transfer via nanotubes between 721.221 EBV transformed B-cells

Figure 1
Membrane proteins can transfer via nanotubes between 721.221 EBV transformed B-cells

The cell to the right is transfected to express the membrane protein GPI-GFP (glycosylphosphatidylinositol-green fluorescent protein) [2] whereas the cell to the left is untransfected. Fluorescent protein was transferred to the untransfected cell at the base of the nanotube. The image is a single optical slice acquired with an inverted confocal microscope (TCS SP2 RS; Leica). Cells were imaged in full culture media (without Phenol Red) at 37°C in 5% CO2. Scale bar, 25 μm.

Figure 1
Membrane proteins can transfer via nanotubes between 721.221 EBV transformed B-cells

The cell to the right is transfected to express the membrane protein GPI-GFP (glycosylphosphatidylinositol-green fluorescent protein) [2] whereas the cell to the left is untransfected. Fluorescent protein was transferred to the untransfected cell at the base of the nanotube. The image is a single optical slice acquired with an inverted confocal microscope (TCS SP2 RS; Leica). Cells were imaged in full culture media (without Phenol Red) at 37°C in 5% CO2. Scale bar, 25 μm.

Bulges and lipid vesicles visible in nanotubes connecting HEK-293T cells

Figure 2
Bulges and lipid vesicles visible in nanotubes connecting HEK-293T cells

Formation of bulges may be related to ‘pearling’ attributed to gradual disruption of the actin cytoskeleton as the tube is stretched [23]. Cells were labelled with the DNA stain Hoechst (blue) and the lipid probe DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate) (red). Fluorescence image is a projection of several optical slices acquired with an inverted confocal microscope (TCS SP2 RS, Leica). DiD staining was carried out by incubating cells in 1 μM DiD, at 37°C for 30 min, washed and left to rest for 24 h before imaging. Hoechst was added to the cells 30 min before imaging. Cells were imaged in full culture media (with Phenol Red) at 37°C and 5% CO2. No fluorescence was observed from unlabelled cells in the same medium. DiD and Hoechst were purchased from Molecular Probes (Eugene, OR, U.S.A.). Scale bar, 25 μm.

Figure 2
Bulges and lipid vesicles visible in nanotubes connecting HEK-293T cells

Formation of bulges may be related to ‘pearling’ attributed to gradual disruption of the actin cytoskeleton as the tube is stretched [23]. Cells were labelled with the DNA stain Hoechst (blue) and the lipid probe DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate) (red). Fluorescence image is a projection of several optical slices acquired with an inverted confocal microscope (TCS SP2 RS, Leica). DiD staining was carried out by incubating cells in 1 μM DiD, at 37°C for 30 min, washed and left to rest for 24 h before imaging. Hoechst was added to the cells 30 min before imaging. Cells were imaged in full culture media (with Phenol Red) at 37°C and 5% CO2. No fluorescence was observed from unlabelled cells in the same medium. DiD and Hoechst were purchased from Molecular Probes (Eugene, OR, U.S.A.). Scale bar, 25 μm.

In addition to the obvious role of axons and dendrites in neuronal cell communication, cellular extensions are in fact observed in a wide variety of species and cell types [7,8]. In plants, for example, it is well established that cells are connected through continuous cytoplasmic bridges, i.e. plasmodesmata [9], which allows flow of small molecules, transport of macromolecules such as proteins and RNA [10] and virus particles [11,12]. In the human immune system, expression of the gene B144/LST1, which is found in the major histocompatibility complex, leads to production of long filopodia, sometimes connecting cells, in a range of cell lines [13]. This gene is endogenously expressed in, for example, dendritic cells and may thus play a role in optimizing these cells' morphology for antigen presentation. Thus, often under specific circumstances, such as chemical activation [14,15], antibody stimulation [16] or overexpression of specific transfected proteins [13,17], cells can use long extensions to establish connections. A network based on long-range multiple contacts, transient or stable, allows additional complexity in the communication between cells in a tissue. By the clustering of distinct ligands or receptors at the tips of multiple extensions, cells could form a complex communication network, in sharp contrast with communication based on chemical gradients, where neighbouring cells would experience the same stimulus [7,8].

It seems clear that a membrane nanotube connection can transport cell surface and cytoplasmic components between cells [1,2]. This can serve to homogenize groups of cells, e.g. in terms of proteins expressed on the cell surface. One speculative reason for this might be that the sharing of proteins between cells is generally useful. For example, detrimental mutations in proteins would not be lethal to a cell that could acquire the protein from connecting cells. Thus detrimental mutations in proteins could persist in case they would lead to better function as a consequence of further mutation.

Balancing any use of an accessible network between cells may be the exploitation of that network by an infectious pathogen. However, a nanotube-based network may exhibit some degree of ‘gate keeping’, preventing mixing of all cytoplasmic material. For example, cytoplasmic material is not transferred between immune cells, despite them being connected through membrane bridges [4].

Advances in experimental procedures to create lipid-based vesicular networks, connected through membrane nanotubes (Figure 3), have proven useful to characterize the physical properties of membrane structures [18,19]. For example, transport properties through tubes [20,21] and along the membrane of tubes [22] have been studied. However, in cell biology, the transient appearance and delicate structure of nanotubes between cells pose particular experimental challenges. Using fluorescence imaging, nanotubes can be characterized in terms of composition, organization, length and duration, and some of the molecules that get transported can be readily observed [1,2]. However, determining whether there are functional consequences of membrane nanotubes connecting immune cells requires innovative experimental approaches. A major limitation is that it may not be possible to observe in vivo membrane nanotubes between immune cells with the currently available technology. Broadly speaking, nanotubular networks are structures of fascinating topology and the concept offers scope for new models of intercellular communication. However, at this point, it is also possible that membrane nanotubes are an in vitro phenomenon. To suppose that membrane nanotubes may be important for immune surveillance is easy; however, proving an important immunological role for nanotubes is difficult and may require new technology.

A constructed network of vesicles connected by membrane nanotubes

Figure 3
A constructed network of vesicles connected by membrane nanotubes

Complex unilamellar lipid bilayer networks can be constructed by micromanipulation methods. During the assembly, it is possible to control the container size and the nanotube length, and various lipid compositions can be used [19,24].

Figure 3
A constructed network of vesicles connected by membrane nanotubes

Complex unilamellar lipid bilayer networks can be constructed by micromanipulation methods. During the assembly, it is possible to control the container size and the nanotube length, and various lipid compositions can be used [19,24].

Lipids, Rafts and Traffic: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by G. Banting (Bristol, U.K.), N. Bulleid (Manchester, U.K.), C. Connolly (Dundee, U.K.), S. High (Manchester, U.K.) and K. Okkenhaug (Babraham Institute, Cambridge, U.K.)

Abbreviations

     
  • DiD

    1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate

  •  
  • HEK-293T

    human embryonic kidney-293T

We thank Owe Orwar and his research group at Chalmers University, Gothenburg, Sweden, for kindly providing Figure 3. This work was supported by the Medical Research Council, the Department of Trade and Industry and the Human Frontier Science Program. B.Ö. was supported by a postdoctoral fellowship from The Wenner-Gren Foundation.

References

References
1
Rustom
A.
Saffrich
R.
Markovic
I.
Walther
P.
Gerdes
H.H.
Science
2004
, vol. 
303
 (pg. 
1007
-
1010
)
2
Önfelt
B.
Nedvetzki
S.
Yanagi
K.
Davis
D.M.
J. Immunol.
2004
, vol. 
173
 (pg. 
1511
-
1513
)
3
Eriksson
M.
Leitz
G.
Fällman
E.
Axner
O.
Ryan
J.C.
Nakamura
M.C.
Sentman
C.L.
J. Exp. Med.
1999
, vol. 
190
 (pg. 
1005
-
1012
)
4
Stinchcombe
J.C.
Bossi
G.
Booth
S.
Griffiths
G.M.
Immunity
2001
, vol. 
15
 (pg. 
751
-
761
)
5
Hochmuth
R.M.
Evans
C.A.
Wiles
H.C.
McCown
J.T.
Science
1983
, vol. 
220
 (pg. 
101
-
102
)
6
Dopheide
S.M.
Maxwell
M.J.
Jackson
S.P.
Blood
2002
, vol. 
99
 (pg. 
159
-
167
)
7
Ramirez-Weber
F.A.
Kornberg
T.B.
Cell (Cambridge, Mass.)
2000
, vol. 
103
 (pg. 
189
-
192
)
8
Rørth
P.
Cell (Cambridge, Mass.)
2003
, vol. 
112
 (pg. 
595
-
598
)
9
Zambryski
P.
J. Cell Biol.
2004
, vol. 
164
 (pg. 
165
-
168
)
10
Kim
M.
Canio
W.
Kessler
S.
Sinha
N.
Science
2001
, vol. 
293
 (pg. 
287
-
289
)
11
Lazarowitz
S.G.
Beachy
R.N.
Plant Cell
1999
, vol. 
11
 (pg. 
535
-
548
)
12
Lucas
W.J.
Gilbertson
R.L.
Annu. Rev. Phytopathol.
1994
, vol. 
32
 (pg. 
387
-
411
)
13
Raghunathan
A.
Sivakamasundari
R.
Wolenski
J.
Poddar
R.
Weissman
S.M.
Exp. Cell Res.
2001
, vol. 
268
 (pg. 
230
-
244
)
14
Ramirez-Weber
F.A.
Kornberg
T.B.
Cell (Cambridge, Mass.)
1999
, vol. 
97
 (pg. 
599
-
607
)
15
Galkina
S.I.
Sud'ina
G.F.
Ullrich
V.
Exp. Cell Res.
2001
, vol. 
266
 (pg. 
222
-
228
)
16
Gupta
N.
DeFranco
A.L.
Mol. Biol. Cell
2003
, vol. 
14
 (pg. 
432
-
444
)
17
De Joussineau
C.
Soule
J.
Martin
M.
Anguille
C.
Montcourrier
P.
Alexandre
D.
Nature (London)
2003
, vol. 
426
 (pg. 
555
-
559
)
18
Evans
E.
Bowman
H.
Leung
A.
Needham
D.
Tirrell
D.
Science
1996
, vol. 
273
 (pg. 
933
-
935
)
19
Karlsson
A.
Karlsson
R.
Karlsson
M.
Cans
A.S.
Strömberg
A.
Ryttsen
F.
Orwar
O.
Nature (London)
2001
, vol. 
409
 (pg. 
150
-
152
)
20
Karlsson
R.
Karlsson
M.
Karlsson
A.
Cans
A.-S.
Bergenholtz
J.
Åkerman
B.
Ewing
A.
Voinova
M.
Orwar
O.
Langmuir
2002
, vol. 
18
 (pg. 
4186
-
4190
)
21
Karlsson
R.
Karlsson
A.
Orwar
O.
J. Phys. Chem.
2003
, vol. 
107
 (pg. 
11201
-
11207
)
22
Davidson
M.
Karlsson
M.
Sinclair
J.
Sott
K.
Orwar
O.
J. Am. Chem. Soc.
2003
, vol. 
125
 (pg. 
374
-
378
)
23
Bar-Ziv
R.
Tlusty
T.
Moses
E.
Safran
S.A.
Bershadsky
A.
Proc. Natl. Acad. Sci. U.S.A.
1999
, vol. 
96
 (pg. 
10140
-
10145
)
24
Karlsson
M.
Sott
K.
Davidson
M.
Cans
A.S.
Linderholm
P.
Chiu
D.
Orwar
O.
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
11573
-
11578
)