The art of cellular communication : tunneling nanotubes bridge the divide The ability of cells to receive , process , and respond to information is essential for a variety of biological processes . This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms . In the latter , most cells do not exist as independent units , but are organized into specialized tissues . Within these functional assemblies , cells communicate with each other in different ways to coordinate physiological processes . Recently , a new type of cell-to-cell communication was discovered , based on de novo formation of membranous nanotubes between cells . These F-actin-rich structures , referred to as tunneling nanotubes -LRB- TNT -RRB- , were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components . The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity . At the same time it emerged that the direct transfer of cargo between cells is a common functional property , suggesting a general role of TNT-like structures in selective , long-range cell-to-cell communication . Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling , it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes . Introduction Communication with the environment is a basic principle of any biological system . With the increasing complexity of higher organisms , cells had to evolve diverse mechanisms to exchange spatial and temporal information crucial for tissue organization and the maintenance of the organism as a whole . Certainly , tissues are not just a loose accumulation of individual cells , but a highly organized population of interacting cells . Thus , in the various types of animal and plant tissues , cell-to-cell communication is important for maintaining a supracellular organization . Probably the best-characterized example of such an organization is given by the animal central nervous system , which is composed of a complex network of interconnected neuron and glial cells . It is widely accepted that the complexity of the brain reflects its enormous number of intercellular links provided by synaptic connectivity -LRB- including gap junctions -RRB- between axons and dendrites . Gap junctions , through which molecules < 1 kDa can penetrate , are also important players in establishing supracellular organization in other tissues containing electrically excitable cells as heart and smooth muscle cells . Furthermore , these proteinaceous channels interconnect cells of the multicellular layer forming epithelial tissues . Finally , cell-to-cell coupling via gap junctions is a general phenomenon during early embryogenesis , where most cells are electrically coupled . Perhaps due to the presence of a thick cell wall , plant cells have evolved thin membrane channels referred to as plasmodesmata -LRB- PD -RRB- to allow direct connections over longer distances -LRB- Baluška et al. 2004a ; Cilia and Jackson 2004 ; Gallagher and Benfey 2005 -RRB- . Because these bridges provide both membrane and cytoplasmic connectivity between cells , plants are regarded as a supracellular assembly -LRB- Baluška et al. 2004b , c -RRB- . PD , in addition to the functions known for gap junctions in animals , facilitate the transfer of ribonucleoparticles , transcription factors and viruses -LRB- Ruiz-Medrano et al. 2004 -RRB- . In 2004 , a new principle of cell-to-cell communication between animal cells , based on the formation of thin membrane channels , was reported -LRB- Rustom et al. 2004 -RRB- . These channels , referred to as tunneling nanotubes -LRB- TNT -RRB- , were initially found in cultures of rat pheochromocytoma -LRB- PC12 -RRB- cells . As PD , they were shown to mediate membrane continuity between connected cells . TNT permit the direct intercellular transfer of organelles , cytoplasmic molecules , and membrane components -LRB- Gerdes et al. 2007 -RRB- . Subsequent studies on other cell types revealed morphologically similar structures directing intercellular transfer of cargo , including pathogens . It thus becomes apparent that nanotubular bridges provide an important and general mechanism of cell-to-cell communication between animal cells . In the following , we summarize the current knowledge and development in the field of nanotubular communication . As awareness of the potential physiological implications of these structures , we will give a short overview on published data describing long and thin cellular protrusions in tissue implicated in cellular communication . Diversity of TNT-like structures TNT connecting PC12 cells are typically 50 -- 200 nm in diameter and can reach lengths up to several cell diameters . They are stretched , interconnecting cells at their nearest distance -LRB- Fig. 1 -RRB- -LRB- Hodneland et al. 2006 ; Rustom et al. 2004 -RRB- . Their structural integrity is sensitive to mechanical stress , chemical fixation , and even to prolonged light exposure . The feature that most strikingly distinguishes TNT from other cellular protrusions is that they hover in the medium and have no contact to the substratum -LRB- Fig. 1a , a1 -RRB- . A structural characterization of TNT between PC12 cells revealed that they contain F-actin as a prominent cytoskeleton element but no microtubules -LRB- Table 1 -RRB- . Further characterization at the ultra-structural level showed a seamless transition of the surface membrane of the TNT with the plasma membranes of the connected cells -LRB- Fig. 1b , b1 , b2 -RRB- . In addition , a continuous membrane with both connected cells was evident in transmission electron micrographs -LRB- Fig. 2a , a1 , a2 -RRB- -LRB- Table 1 -RRB- . These observations , together with the documentation of a limited lateral diffusion of fluorescent membrane proteins occurring between the plasma membranes of TNT-connected cells , led to the proposal that TNT mediate intercellular membrane continuity -LRB- Fig. 3a -RRB- -LRB- Gerdes et al. 2007 ; Rustom et al. 2004 -RRB- . Fig. 1Architecture of TNT between cultured PC12 cells . -LRB- a -RRB- 3D fluorescence image -LRB- -LRB- x − y -RRB- - maximum projection of 40 consecutive 400 nm sections -RRB- of a wheat germ agglutinin-stained TNT connecting two live PC12 cells . -LRB- a1 -RRB- -LRB- x − z -RRB- - projection in the plane of the TNT indicated in -LRB- a -RRB- . -LRB- b -RRB- Scanning electron micrograph -LRB- SEM -RRB- showing the ultra-structure of a TNT between two PC12 cells . The boxed areas are shown as higher magnification images -LRB- b1 , b2 -RRB- . Modified from Rustom et al. -LRB- 2004 -RRB- Science 303:1007 -- 1010 . Scale bars , a , a1 , b , 5 μm ; b1 , b2 , 500 nmTable 1TNT-like structures in vitroaCell type TNT-like structures Cytoskeletal components Membrane continuity / `` open-ended '' Cargo PC12 cells -LRB- Rustom et al. 2004 -RRB- F-actin , myosin Va -LRB- + -RRB- b Endosome-related organelles , lipid-anchored proteins -LRB- EGFP-f -RRB- , EGFP-actin NRK cells -LRB- Rustom et al. 2004 -RRB- c -LRB- F-actin , myosin Va -RRB- c ND Endosome-related organelles EBV-transformed human B cell line -LRB- 721.221 -RRB- -LRB- Önfelt et al. 2004 -RRB- ND ND GPI-GFP , -LRB- HLA-Cw6-GFP , in coculture with human peripheral blood Natural Killer cells -RRB- d Between neonatal rat CM and adult human EPC -LRB- Koyanagi et al. 2005 -RRB- ND ND Mitochondria , soluble proteins -LRB- GFP -RRB- Primary cultures of rat astrocytes -LRB- Zhu et al. 2005 -RRB- F-actin , myosin Va ND ND DC -LRB- Watkins and Salter 2005 -RRB- ND -LRB- + -RRB- e Calcium fluxes , surface receptors -LRB- HLA-A , B , C class I MHC -RRB- d THP-1 monocytes -LRB- Watkins and Salter 2005 -RRB- F-actin -LRB- + -RRB- e Calcium fluxes , the fluid phase marker lucifer yellow , surface receptors -LRB- HLA-A , B , C class I MHC -RRB- d Between DC and THP-1 monocytes -LRB- Watkins and Salter 2005 -RRB- ND -LRB- + -RRB- e Calcium fluxes Human monocyte-derived macrophages -LRB- bridges with diameter ≥ 0.7 μm -RRB- -LRB- Önfelt et al. 2006 -RRB- F-actin , microtubules -LRB- + -RRB- f Mitochondria , endosome-related organelles , lysosomes Human monocyte-derived macrophages -LRB- nanotubes with diameter < 0.7 μm -RRB- -LRB- Önfelt et al. 2006 -RRB- F-actin ND Surfing Mycobacterium bovis bacillus Calmette-Guérin Cos-1 , XC and HEK 293 cells -LRB- Sherer et al. 2007 -RRB- F-actin -LRB- − -RRB- b MLV Jurkat T cells -LRB- Sowinski et al. 2008 -RRB- F-actin -LRB- − -RRB- b , e HIV-1 protein Gag aOnly those publications that fulfill at least one of the listed criteria are shownbAccessed by electron microscopycGurke , S. , Barroso , J. , Bukoreshtliev , N. , Gerdes , H.-H. , unpublished datadThese molecules were shown to localize in TNT-like structures , but their intercellular transfer was not proveneAccessed by the measurement of calcium fluxesfProposed from the observation of a seamless transition between microtubules of the bridge with microtubular networks of both connected cellsND : not determinedFig . 2Transmission electron micrographs -LRB- TEM -RRB- showing the ultra-structure of distinct TNT-like bridges in different cell types . -LRB- a -RRB- Open-ended TNT connecting two PC12 cells reconstructed from images of two consecutive 80 nm sections . The boxed areas are shown as higher magnification images -LRB- a1 , a2 -RRB- . A continuous membrane is observed between the nanotube and the plasma membrane of the two connected cells . Modified from Rustom et al. -LRB- 2004 -RRB- Science 303:1007 -- 1010 . -LRB- b -RRB- Close-ended TNT-like bridge connecting two T cells and displaying a junctional border , reconstructed from images of 13 consecutive 60 nm sections . The boxed areas are shown as higher magnification images -LRB- b1 , b2 -RRB- . The nanotube formed by one cell -LRB- b2 -RRB- protrudes into an invagination -LRB- arrowhead -RRB- of the connected cell -LRB- b1 -RRB- . Modified from Sowinski et al. -LRB- 2008 -RRB- Nat Cell Biol 10:211 -- 219 . Scale bars , a , b , 1 μm ; a1 , a2 , b1 , b2 , 500 nmFig . 3Schematic representations of three distinct nanoscaled cellular protrusions and proposed modes of cell-to-cell communication . -LRB- a -RRB- A TNT-mediating membrane continuity between cells . -LRB- a1 -RRB- Organelles like endocytic vesicles and mitochondria are transported uni-directionally between cells by an actin-dependent mechanism . -LRB- b -RRB- Nanotubular bridge between cells displaying a junctional border . -LRB- b1 -RRB- Distinct viral particles are transported either at the surface of the nanotube by a receptor-dependent mechanism using actin retrograde flow or inside the cellular nanotube by an actin-dependent mechanism . -LRB- c -RRB- Cellular nanotube -LRB- cytoneme -RRB- extending toward a target cell by chemotaxis . -LRB- c1 -RRB- Signaling molecules secreted by the target cell are proposed to be endocytosed by a receptor-mediated mechanism at the tip of the cytoneme and transported in a retrograde manner toward the cell body of the receiving cell . The arrows -LRB- a -- c -RRB- indicate the direction of transfer Based on the morphological criteria defined for PC12 cells , similar TNT-like connections were subsequently identified for several permanent cell lines and primary cultures -LRB- for a comprehensive overview see -LRB- Gerdes et al. 2007 -RRB- -RRB- -LRB- Table 1 -RRB- . TNT-like bridges between dendritic cells -LRB- DC -RRB- -LRB- Watkins and Salter 2005 -RRB- and perhaps also those connecting neonatal rat cardiomyocytes -LRB- CM -RRB- and adult human endothelial progenitor cells -LRB- EPC -RRB- -LRB- Koyanagi et al. 2005 -RRB- , appear to be most closely related to the TNT described for PC12 cells . The flow of cytoplasmic molecules suggests membrane continuity between connected cells -LRB- Table 1 -RRB- . This contrasts the TNT-like bridges between T cells , which have been characterized lately in considerable detail -LRB- Sowinski et al. 2008 -RRB- . The observed T-cell nanotubes have an average length of ∼ 20 μm , a diameter of 180 -- 380 nm , and are not tethered to the substratum . Moreover , as in PC12 cells , only F-actin but not microtubules were detected in T-cell nanotubes -LRB- Fig. 3 -RRB- . However , despite the overall similarity in architecture and capacity to facilitate intercellular transfer of cargo between nanotubes of PC12 cells , DC , CM/EPC and T-cells , the latter do not mediate membrane continuity between connected cells -LRB- Fig. 2b , b1 , b2 -RRB- -LRB- Table 1 -RRB- . This was concluded from the observation that nanotubes between T-cells -LRB- i -RRB- do not facilitate the transfer of cytoplasmic molecules , -LRB- ii -RRB- do not permit free diffusion of fluorescent plasma membrane components and -LRB- iii -RRB- display a junctional border between the nanotube and the connected T-cell at the ultra-structural level -LRB- Fig. 2b1 -RRB- . Accordingly , they were classified as `` not open-ended '' cellular nanotubes -LRB- Fig. 3b -RRB- . Other identified close-ended nanotubular bridges that share some morphological features with TNT are murine leukemia virus-induced `` filopodial bridges '' -LRB- Sherer et al. 2007 -RRB- -LRB- Table 1 -RRB- . Interestingly , it has been shown that different classes of nanotubes exist even within a single cell type . In the case of macrophages , not only thin , F-actin-containing bridges similar to other TNT-like structures were detected , but also a thicker type of cellular connection -LRB- ≥ 0.7 μm diameter -RRB- , which contained both F-actin and microtubules -LRB- Önfelt et al. 2006 -RRB- . This thicker type of connection was suggested to be open ended due to a seamless transition of the microtubules inside the bridge with the microtubular networks of both connected cells -LRB- Önfelt et al. 2006 -RRB- -LRB- Table 1 -RRB- . Thin , and thicker microtubule-containing connections were also found between prostate cancer cells -LRB- Vidulescu et al. 2004 -RRB- . Finally , another type of long and thin F-actin-containing cellular protrusions that share striking features with TNT-like intercellular bridges , are cytonemes . These membrane nanotubes were discovered in Drosophila imaginal wing discs , emanating from the periphery of the columnar cell sheet toward the signaling center associated with the anterior/posterior border -LRB- Ramírez-Weber and Kornberg 1999 -RRB- . They are thought to extend toward the target cells by chemotaxis -LRB- Ramírez-Weber and Kornberg 1999 -RRB- -LRB- Fig. 3c -RRB- to accomplish the receptor-mediated uptake of the secreted morphogen Decapentaphlegic -LRB- Dpp -RRB- during spatial patterning -LRB- Hsiung et al. 2005 -RRB- . Once endocytosed , the morphogen is thought to be delivered to the cell body by retrograde transport along the cytoneme -LRB- Fig. 3c1 -RRB- . Thus , these structures are proposed to fulfill an important task in long-range cell-to-cell signaling during embryonic development . In contrast to TNT-like structures , cytonemes have not been shown to physically bridge cells . Nevertheless , functionally similar structures , emanating from tracheal cells in response to Branchless -LRB- Bnl -RRB- - Fibroblast Growth Factor -LRB- FGF -RRB- signaling , appear to do so -LRB- Sato and Kornberg 2002 -RRB- . The identification of TNT-like structures in cell cultures has been mainly based on the morphological criteria defined for PC12 cells -LRB- Rustom et al. 2004 -RRB- . In some cases , a proper comparison was hindered by their limited characterization . Nonetheless , heterogeneity regarding formation , structure , and functional properties across cell types and even within a given cell type has emerged -LRB- Table 1 -RRB- . Certainly , more detailed information is necessary for a proper classification of all these structures and thus , as things are now , we shall refer to them as TNT-like structures , irrespective of whether membrane continuity was observed . Formation of TNT-like structures TNT-like structures form de novo between cells on a time-scale of several minutes by apparently two distinct mechanisms . With respect to the first mechanism , initial studies on PC12 cells showed that TNT could be established by a seemingly directed outgrowth of swaying filopodia-like protrusion -LRB- s -RRB- toward a neighboring cell . Once contact is made , a single dilated and bended bridge is often observed along with the degeneration of remainder protrusions . This bended structure is then remodeled into a straight and thin bridge showing the characteristic morphology of typical TNT -LRB- Rustom et al. 2004 -RRB- . One can speculate that the seemingly directed filopodia outgrowth preceding TNT formation is under control of chemotactic guidance . Evidence for this is provided for related processes like the formation of cytonemes toward a Bnl-FGF gradient in in vitro cultures of Drosophila cells -LRB- Ramírez-Weber and Kornberg 1999 -RRB- -LRB- Fig. 3c -RRB- or murine leukemia virus -LRB- MLV -RRB- - induced nanotubular bridges emanating from non-infected fibroblast cells toward infected cells -LRB- Sherer et al. 2007 -RRB- . The observation that TNT-like bridges also emerge when attached PC12 -LRB- Rustom et al. 2004 -RRB- or immune cells -LRB- Önfelt et al. 2004 , 2006 ; Sowinski et al. 2008 -RRB- depart from each other , led to the proposal of a second mechanism of TNT formation -LRB- Gerdes et al. 2007 ; Önfelt et al. 2004 -RRB- , which a priori excludes any dependence on chemotactic guidance of filopodia . Subsequent detailed studies on T cells revealed that cell-to-cell interaction for at least a few minutes before dislodging is required for successful nanotubular bridge formation -LRB- Sowinski et al. 2008 -RRB- . It is of note that , in contrast to PC12 cells , a filopodium-dependent mechanism of nanotubular bridge formation between immune cells was not reported to date -LRB- Önfelt et al. 2004 , 2006 ; Sowinski et al. 2008 -RRB- . Actin polymerization is thought to be a key event for both mechanisms of TNT formation -LRB- Gerdes et al. 2007 -RRB- . It drives filopodia outgrowth important in the first mechanism and is most probably important for the stabilization of TNT-like structures emerging by the second mechanism . In support of this , TNT formation is not observed in the presence of actin-depolymerizing drugs -LRB- Rustom et al. 2004 -RRB- , and TNT-like structures between T cells only form if the cells diverge with a speed below that of processes driven by actin polymerization -LRB- Cameron et al. 1999 ; Sowinski et al. 2008 -RRB- . Zhu et al. -LRB- 2005 -RRB- obtained further evidence that actin polymerization and TNT formation are linked by demonstrating their concurrent induction by H2O2 in primary cultures of rat astrocytes . TNT-like structures have a dynamic nature leading to only transient bridges as observed in in vitro cultures . The nanotubular bridges between T cells -LRB- Sowinski et al. 2008 -RRB- , PC12 cells or normal rat kidney -LRB- NRK -RRB- cells -LRB- Bukoreshtliev , N. , Gerdes , H.-H. , unpublished data -RRB- have variable lifetimes , ranging from a few minutes to less than 60 min for the former and even up to several hours for the latter two . These considerable differences in lifetime , even at the cellular level , may reflect the existence of different subclasses of TNT-like structures . It is interesting to speculate that the fusion of the nanotube with the plasma membrane of the connected cell is part of a time-dependent maturation step leading to such heterogeneity . Studies on model artificial membrane tubes such as those created by pulling tethers from synthetic lipid vesicles or cellular plasma membrane provide a complementary approach to obtain mechanistic insights into the formation of TNT-like structures . The morphology of such nanotubes resembles that of TNT-like structures and it seems plausible that the same physical laws govern the formation and architecture of both structures . The exploitation of such in vitro models revealed that the elongation of plasma membrane nanotubes requires a membrane flow from the cell plasma membrane into the growing tube . It is suggested that cells maintain a membrane reservoir -LRB- e.g. ruffles , invaginations -RRB- , controlled by the cytoskeleton , to provide a buffer against membrane tension over several micrometers of tube elongation -LRB- Raucher and Sheetz 1999 ; Sun et al. 2005 -RRB- . Thus , the available membrane reservoir may restrict the number , total length , and lifetime of TNT-like connections for a given cell . TNT-like structures are conduits for the delivery of cargo An obvious advantage of establishing direct bridges between cells is an improvement in selective communication even over long distances . In agreement with this assumption , the common feature of all TNT-like structures characterized in more detail , is the transfer of cargo between connected cells -LRB- Table 1 -RRB- . Cellular components Direct evidence for the intercellular exchange of cargo via TNT-like structures was obtained by video-microscopic studies . By employing fluorescent dyes , organelles belonging to the endosomal/lysosomal system -LRB- Rustom et al. 2004 -RRB- -LRB- Gurke , S. , Gerdes , H.-H. , unpublished data -RRB- as well as mitochondria -LRB- Koyanagi et al. 2005 -RRB- were shown to traffic uni-directionally along TNT-like structures between cells over long distances -LRB- Fig. 3a1 -RRB- -LRB- Table 1 -RRB- . The occurrence of an intercellular transfer of organelles raises the question as to what kind of information is transferred and how this information is integrated in the target cell . In the case of the endosomal system , early endocytic vesicles are particularly interesting candidates for TNT-mediated delivery . These organelles are one of the major reloading points for a variety of signaling complexes resulting from the endocytosis of activated cell surface receptors -LRB- Miaczynska et al. 2004 -RRB- . Once delivered to the target cells , the transferred endocytic vesicles are able to fuse with their counterparts -LRB- Rustom et al. 2004 -RRB- , and provide a potential way to integrate information . In essence , the transfer of endocytic organelles propagates signaling information from the single cell to a larger community , which may lead to coordinated cell behavior . Mitochondria were shown to transfer via TNT from neonatal rat CM to adult human EPC and it was suggested that this event could contribute to the acquisition of a cardiomyogenic phenotype by the progenitor cells -LRB- Koyanagi et al. 2005 -RRB- -LRB- Fig. 4 -RRB- . Moreover , the active transfer of this organelle from adult stem cells and somatic cells to mammalian cells with non-functional mitochondria was shown to rescue aerobic respiration in the latter -LRB- Csordás 2006 ; Spees et al. 2006 -RRB- . The obtained data are consistent with a TNT-related delivery , although alternative mechanisms were not excluded . Fig. 4Emerging physiological implications of TNT-like structures In addition to the transfer of organelles , plasma membrane components such as lipid-anchored proteins can laterally transfer along the TNT-like bridge into the plasma membrane of connected cells . This was demonstrated for both the inner and outer leaflets of the plasma membrane by using glycosylphosphatidylinositol conjugated to GFP -LRB- GPI -RRB- - GFP -LRB- Önfelt and Davis 2004 ; Önfelt et al. 2004 -RRB- and the farnesylation signal of c-Ha-Ras fused to the C-terminus of EGFP -LRB- EGFP-f -RRB- -LRB- Rustom et al. 2004 -RRB- , respectively -LRB- Table 1 -RRB- . Such a transport is consistent with membrane continuity between connected cells . Surface receptors could also be transferred directly by lateral diffusion in the plane of the cell surface , in addition to an endosome-dependent delivery . Interestingly , the transfer of membrane proteins between myeloid cells was found to involve scavenger receptors -LRB- Dr. R. D. Salter , Univ. of Pittsburgh , personal communication -RRB- . It is of note that major histocompatibility complex -LRB- MHC -RRB- class I receptors were detected at the entire length of TNT-like bridges connecting immune cells -LRB- Önfelt et al. 2004 ; Watkins and Salter 2005 -RRB- . This points to a key role of TNT in a faster and more efficient presentation of antigens , in particular at the immunological synapse -LRB- Groothuis et al. 2005 ; Williams et al. 2007 -RRB- -LRB- Fig. 4 -RRB- , defined as a highly specialized interface between immune cells -LRB- Norcross 1984 ; Paul and Seder 1994 -RRB- . In this case , the lateral transfer of only a few molecules can fulfill important roles in a coordinated immune response . Interestingly , in analogy to TNT , membrane continuity was found at the immunological synapse -LRB- Stinchcombe et al. 2001 -RRB- . The characterized `` membrane bridges '' had a diameter of 50 -- 95 nm as observed by electron microscopy -LRB- Stinchcombe et al. 2001 -RRB- . Finally , the proposed model of membrane continuity suggests a cytoplasmic bridge in analogy to PD , which permits the free flow of soluble molecules up to a certain size limit defined by the free space along the interior of the nanotube . In support of this view , EGFP-actin was transferred between TNT-connected PC12 cells -LRB- Rustom et al. 2004 -RRB- , GFP between CM and EPC -LRB- Koyanagi et al. 2005 -RRB- , and the fluid phase marker Lucifer yellow between THP-1 monocytes -LRB- Watkins and Salter 2005 -RRB- -LRB- Table 1 -RRB- . Notably , both GFP and the small dye molecule calcein , with a molecular weight of 400 Da , were apparently impeded to diffuse passively between TNT-connected PC12 cells -LRB- Rustom et al. 2004 -RRB- . Thus , TNT-like structures , like PD , may have a variable size exclusion limit depending on the cell type -LRB- Gerdes et al. 2007 -RRB- . With regard to soluble markers , certainly the most physiologically relevant finding was the TNT-dependent propagation of calcium signals between THP-1 monocytes as well as from DC to THP-1 monocytes -LRB- Watkins and Salter 2005 -RRB- -LRB- Table 1 -RRB- . Stimulation by contact or exogenous soluble stimulators of both DC and THP-1 monocytes led to a calcium flux that propagated through a network of TNT-connected cells up to 500 μm away from the point of stimulation . The elicited calcium wave propagated uni-directional with an initial speed of 35 μm/s that slowed down rapidly to 10 -- 15 μm/s -LRB- Watkins and Salter 2005 -RRB- . This rather low and rapidly decreasing speed supports a TNT-mediated signal spread along the cellular network , which is driven by a chemical gradient rather than an action potential . Such a view also corroborates a TNT-mediated membrane continuity providing a cytoplasmic bridge between connected cells -LRB- Table 1 -RRB- . Most remarkably , the transfer of calcium ions through TNT led to the flattening and membrane extension of the receiving cells , as occurs during physiological responses in preparation for phagocytosis -LRB- Watkins and Salter 2005 -RRB- . Thus , this study provided the first evidence for functional connectivity accomplished by TNT and suggests a crucial role of these structures in immune defense -LRB- Fig. 4 -RRB- . Pathogens An emerging topic is the efficient spread of pathogens , like bacteria and mammalian viruses , along cell membrane protrusions -LRB- Figs. 3 , 4 -RRB- . By using established or induced nanotubular connections between cells , pathogens circumvent the rate-limiting step of diffusion and efficiently accelerate their spreading -LRB- Sherer et al. 2007 ; Sowinski et al. 2008 -RRB- . Among the viruses shown to exploit cellular protrusions to transfer from cell-to-cell are the vaccinia virus -LRB- Cudmore et al. 1995 , . 1996 -RRB- , pseudorabies virus -LRB- Favoreel et al. 2005 , 2006 -RRB- , the herpes simplex virus -LRB- La Boissière et al. 2004 -RRB- , MLV -LRB- Sherer et al. 2007 -RRB- and human immunodeficiency virus type 1 -LRB- HIV-1 -RRB- -LRB- Sowinski et al. 2008 -RRB- . In the following , we will focus on the latter two , which were characterized in much detail and gave rise to two different mechanisms of virus transmission . Sowinski et al. -LRB- 2008 -RRB- proposed membrane nanotubes between T cells as a new route for HIV-1 transmission -LRB- Fig. 3b1 -RRB- , in addition to cellular contact points known as virological synapses -LRB- Jolly et al. 2004 ; Jolly and Sattentau 2004 ; Sol-Foulon et al. 2007 -RRB- . Using a recombinant HIV-1 expressing Gag-GFP , they demonstrated its uni-directional move through innate nanotubular bridges and subsequent transmission into connected T cells -LRB- Sowinski et al. 2008 -RRB- -LRB- Table 1 -RRB- . Furthermore , the requirement of cell surface receptors for viral transmission implied , in agreement with ultra-structural data , a junctional border between the nanotube and the connected T cell . It should be emphasized that the transmission of HIV-1 through nanotubular bridges can minimize the exposure of the virus to extracellular antibodies or complement . However , a nanotube-dependent spread may open new avenues in the development of antiviral drugs . A different , even though nanotube-dependent , mechanism of transmission was reported for MLV -LRB- Sherer et al. 2007 -RRB- . The cell-to-cell transmission of MLV was directly visualized by infecting cultured cells with a recombinant virus expressing the viral capsid protein Gag and the envelope protein Env as a fusion construct with fluorescent proteins . MLV induces the outgrowth of thin and long filopodia from uninfected cells toward infected cells -LRB- Sherer et al. 2007 -RRB- . These filopodia are proposed to stably anchor to the cell body of an infected cell by viral envelope glycoprotein -LRB- Env -RRB- / receptor interactions . Subsequently , MLV moves along the outer surface of these nanotubular bridges to reach the cell body of target cells , where it finally enters -LRB- Sherer et al. 2007 -RRB- -LRB- Fig. 3b1 -RRB- . In addition to viruses , bacteria were reported to surf along TNT-like structures connecting macrophages -LRB- Önfelt et al. 2006 -RRB- -LRB- Table 1 -RRB- . Mechanism of transfer It is possible that more than one transfer mechanism for shipping cargo via TNT-like bridges has evolved considering their structural diversity and the multitude of transferred cargo . For organelles and viruses , an F-actin-dependent , uni-directional transport prevails -LRB- Fig. 3 -RRB- , with the exception of a microtubule-dependent , bi-directional movement of organelles in thick bridges connecting macrophages -LRB- Önfelt et al. 2006 -RRB- . In the case of endocytic organelles , an acto-myosin-dependent transport system is presumed -LRB- Fig. 3a1 -RRB- due to the presence of the barbed-end directed actin-specific motor myosin Va in TNT -LRB- Rustom et al. 2004 ; Zhu et al. 2005 -RRB- -LRB- Bukoreshtliev , N. , Gerdes , H.-H. , unpublished data -RRB- -LRB- Table 1 -RRB- partially co-localizing with the respective organelles -LRB- Rustom et al. 2004 -RRB- . This model is supported by the low transport velocity in the range of actin-dependent transport -LRB- Rustom et al. 2004 -RRB- and implies that the F-actin fibers inside the bridge have the same polarity . Also HIV-1 particles were suggested to move in an actin-dependent manner through nanotubular bridges -LRB- Sowinski et al. 2008 -RRB- -LRB- Fig. 3b1 -RRB- . Interestingly , even in the case of MLV surfing along filopodia bridges with a speed ∼ 7 times slower as compared to that of HIV-1 within the bridges , an acto-myosin transfer mechanism appears to be in place -LRB- Sherer et al. 2007 -RRB- . This results in the uni-directional movement of all viral particles from infected to target cells , presumably using myosin II controlled retrograde flow of actin and cognate Env-receptor interactions -LRB- Fig. 3b1 -RRB- . In contrast to organelles and viruses , the uni-directional propagation of calcium waves is likely to be driven by a chemical gradient rather than an active transport mechanism . Passive diffusion is likely to control the transfer of other small cytoplasmic molecules and also some plasma membrane components , if membrane continuity is present . Regarding the modality by which shipped cargo enters the target cell , two different scenarios are conceivable . In the case of membrane continuity as observed in PC12 cells , an open-ended transport along cytoskeleton elements without border crossing is envisaged , consistent with the observed uniform movement of transferred organelles -LRB- Rustom et al. 2004 -RRB- . In analogy to PD , gating mechanisms may exist to control this open-ended transport -LRB- Gerdes et al. 2007 -RRB- . In the case of close-ended cell-to-cell bridges containing junctional borders , as are the innate nanotubes connecting T cells and the MLV-induced cellular bridges , the cargo has to traverse the plasma membrane boundary . This is consistent with the finding that a receptor-dependent transmission of HIV-1 Gag could be blocked by the HIV-1 fusion inhibitor T20 -LRB- Sowinski et al. 2008 -RRB- . Ultra-fine cellular extensions in tissue Does intercellular communication mediated by TNT-like structures play a physiological role in tissue ? This is probably the most interesting and important question that needs to be addressed in the near future . The absence of a TNT-specific marker does not allow an explicit answer to this question yet . However , studies employing cell cultures already point to important physiological implications of TNT-like structures in intercellular communication -LRB- reviewed in Gerdes et al. -LRB- 2007 -RRB- -RRB- -LRB- Fig. 4 -RRB- . It could be argued that cell culture models are rough representations of physiological systems and that TNT-like structures are the sole result of stress conditions in this in vitro situation . However , the widely documented richness of cellular protrusions in tissue implicated in intercellular communication -LRB- Rørth 2003 -RRB- -LRB- Table 2 -RRB- disagrees with such a possibility and instead supports an in vivo function . Cellular extensions have been documented in diverse tissues and during various physiological processes such as embryogenesis , stem cell differentiation , cell migration , and wound healing . Table 2Selected examples of cell protrusions in tissueProtrusions Tissue Cytoskeletal components Cargo Length -LSB- μm -RSB- Diameter -LSB- nm -RSB- Growth speed -LSB- μm/min -RSB- Thin , elongated , active filopodia and lamellipodia Primary mesenchyme sea urchin embryos -LRB- Gustafson and Wolpert 1967 ; Miller et al. 1995 -RRB- F-actinaND 80a200 -- 400b25a Cytonemes Drosophila wing imaginal discs -LRB- Ramírez-Weber and Kornberg 1999 -RRB- F-actin ND < 700 200 15 Cytoneme-like Mouse limb bud cells -LRB- Ramírez-Weber and Kornberg 1999 -RRB- ; Drosophila wing imaginal discs -LRB- Jacinto et al. 2000 -RRB- F-actinbND 10bND 1bApical peripodial extensions Drosophila wing/eye imaginal discs -LRB- Gibson and Schubiger 2000 -RRB- Microtubules Mitochondriac 5 -- 30 ND ND Myopodia Drosophila embryonic muscle cells -LRB- Ritzenthaler et al. 2000 ; Ritzenthaler and Chiba 2003 -RRB- F-actin ND 40 ND ND Myopodia-like Mouse transverse muscle of thorax -LRB- Misgeld et al. 2002 -RRB- ; Xenopus -LRB- Kullberg et al. 1977 -RRB- ND ND 10dND ND Cellular extensions Drosophila eye imaginal discs -LRB- Chou and Chien 2002 -RRB- F-actin Scabrous vesicles 40 ND ND Apical and lateral cell protrusion Drosophila wing imaginal discs -LRB- Demontis and Dahmann 2007 -RRB- F-actin GFP-Rab5c , GFP-Tkvc1 -- 10 200 ND aData from Miller et al. -LRB- 1995 -RRB- bData from Jacinto et al. -LRB- 2000 -RRB- cThese molecules were shown to localize in cell protrusions , but their intercellular transfer was not provendData from Misgeld et al. -LRB- 2002 -RRB- ND : not determined The formation of cellular extensions -LRB- `` pseudopods '' -RRB- during embryogenesis became first evident by studying sea urchin morphogenesis -LRB- Gustafson and Wolpert 1967 ; Miller et al. 1995 -RRB- , suggesting a function of these structures in force generation or cell recognition . During subsequent studies over the last decades , a variety of similar cellular extensions were found to be associated with developmental processes . Interestingly , most of them appear to be actin-rich as TNT-like structures . The Drosophila wing imaginal disc provides an attractive system for studying cell protrusions within a developing tissue . The first described example of such protrusions were the F-actin-containing cytonemes extending from the periphery of the columnar cell sheet toward the signaling center within this layer -LRB- Ramírez-Weber and Kornberg 1999 -RRB- . This discovery inspired further studies describing other cellular protrusions in the wing disc extending not only within -LRB- Chou and Chien 2002 ; Demontis and Dahmann 2007 -RRB- but also between cell layers -LRB- Demontis and Dahmann 2007 ; Gibson and Schubiger 2000 -RRB- . In a very recent study on live wing discs , Demontis and Dahmann -LRB- 2007 -RRB- observed two new types of cellular extensions , i.e. lateral protrusions interconnecting distant cells of the columnar epithelium , and protrusions of the apical surface connecting to the squamous epithelium . Furthermore , ectopically expressed GFP-actin and the early endosome marker GFP-Rab 5 were detected inside these protrusions , resembling the finding made for TNT-like structures between cultured cells . These different protrusions may be involved in the intercellular signaling necessary for growth and patterning of wing imaginal discs -LRB- Demontis and Dahmann 2007 -RRB- . Thin , actin-based membrane extensions were also found to be crucial for embryonic dorsal closure in Drosophila -LRB- Jacinto et al. 2000 -RRB- . Data obtained by confocal imaging of living fly embryos suggest that these long protrusions participate in both the mechanics of epithelial adhesion and in the search for a correct partner within the opposing epithelium . Likewise , related fundamental processes such as gastrulation , neural crest closure -LRB- Bard 1992 -RRB- , and wound healing -LRB- Wood et al. 2002 -RRB- seem to involve similar actin-rich membrane extensions . Other examples of actin-based cellular extensions during development are the so-called myopodia in Drosophila embryonic muscle cells that can be observed in whole mount . These dynamic postsynaptic microprocesses , with lengths up to 40 μm and highly sensitive to photoillumination , cluster at the site of motoneuron innervation while interacting with presynaptic filopodia . It was proposed that they play a role in the process of synaptic target recognition by contributing to direct long-distance cellular communication -LRB- Ritzenthaler et al. 2000 ; Ritzenthaler and Chiba 2003 -RRB- . Importantly , similar structures have been described in Xenopus -LRB- Kullberg et al. 1977 -RRB- and mouse -LRB- Misgeld et al. 2002 -RRB- . Finally , long , thin cellular extensions are also associated with guided cellular migration during development -LRB- Ribeiro et al. 2002 ; Sato and Kornberg 2002 -RRB- , immune defense such as the migration of macrophages through the endothelial cell layer -LRB- Blue and Weiss 1981 -RRB- and tumor cell invasion -LRB- Vignjevic and Montagnac 2008 -RRB- . Certainly , there are many more examples of long cellular nanotubes in tissue involved in cellular communication besides the ones listed here -LRB- Table 2 -RRB- . This suggests that extensions in tissue are a rather common feature and that cells in culture preserve the ability to form similar , physiologically relevant structures . But , an important question remains : Do TNT-like structures , observed in cell culture models , have mechanistically and/or functionally related counterparts in live tissue ? Furthermore , with respect to TNT , which have been found to mediate membrane continuity between cultured cells , it will be of foremost interest to find out , if this type of connection also exists in vivo .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

tnt
intercellular transfer
cytoneme
viral transmission