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 ( including gap junctions ) 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 . 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 ( pd ) to allow direct connections over longer distances ( baluka et al . because these bridges provide both membrane and cytoplasmic connectivity between cells , plants are regarded as a supracellular assembly ( baluka et al . pd , in addition to the functions known for gap junctions in animals , facilitate the transfer of ribonucleoparticles , transcription factors and viruses ( ruiz - medrano et  al . 2004 ) . in 2004 , a new principle of cell - to - cell communication between animal cells , based on the formation of thin membrane channels , was reported ( rustom et al . 2004 ) . these channels , referred to as tunneling nanotubes ( tnt ) , were initially found in cultures of rat pheochromocytoma ( pc12 ) cells . tnt permit the direct intercellular transfer of organelles , cytoplasmic molecules , and membrane components ( gerdes et  al . 2007 ) . 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 . tnt connecting pc12 cells are typically 50200  nm in diameter and can reach lengths up to several cell diameters . they are stretched , interconnecting cells at their nearest distance ( fig .  1 ) ( hodneland et  al . 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 ( fig .  1a , a1 ) . a structural characterization of tnt between pc12 cells revealed that they contain f - actin as a prominent cytoskeleton element but no microtubules ( table  1 ) . 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 ( fig . in addition , a continuous membrane with both connected cells was evident in transmission electron micrographs ( fig . 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 ( fig . ( a ) 3d fluorescence image ( ( xy)-maximum projection of 40 consecutive 400  nm sections ) of a wheat germ agglutinin - stained tnt connecting two live pc12 cells . ( a1 ) ( xz)-projection in the plane of the tnt indicated in ( a ) . ( b ) scanning electron micrograph ( sem ) showing the ultra - structure of a tnt between two pc12 cells . scale bars , a , a1 , b , 5  m ; b1 , b2 , 500  nmtable  1tnt - like structures in vitrocell type tnt - like structures cytoskeletal components membrane continuity/  open - ended  cargo pc12 cells ( rustom et al . 2004 ) f - actin , myosin va ( + ) endosome - related organelles , lipid - anchored proteins ( egfp - f ) , egfp - actin nrk cells ( rustom et al . 2004)(f - actin , myosin va)nd endosome - related organelles ebv - transformed human b cell line ( 721.221 ) ( nfelt et al . 2004 ) nd nd gpi - gfp , ( hla - cw6-gfp , in coculture with human peripheral blood natural killer cells)between neonatal rat cm and adult human epc ( koyanagi et al . 2005 ) nd nd mitochondria , soluble proteins ( gfp ) primary cultures of rat astrocytes ( zhu et al . 2005 ) f - actin , myosin va nd nd dc ( watkins and salter 2005 ) nd ( + ) calcium fluxes , surface receptors ( hla - a , b , c class i mhc)thp-1 monocytes ( watkins and salter 2005 ) f - actin ( + ) calcium fluxes , the fluid phase marker lucifer yellow , surface receptors ( hla - a , b , c class i mhc)between dc and thp-1 monocytes ( watkins and salter 2005 ) nd ( + ) calcium fluxes human monocyte - derived macrophages ( bridges with diameter 0.7  m ) ( nfelt et al . 2006 ) f - actin , microtubules ( + ) mitochondria , endosome - related organelles , lysosomes human monocyte - derived macrophages ( nanotubes with diameter < 0.7  m ) ( nfelt et al . 2006 ) f - actin nd surfing mycobacterium bovis bacillus calmette - gurin cos-1 , xc and hek 293 cells ( sherer et al . 2008 ) f - actin ( )hiv-1 protein gag only those publications that fulfill at least one of the listed criteria are shownaccessed by electron microscopygurke , s. , barroso , j. , bukoreshtliev , n. , gerdes , h .- h . , unpublished datathese molecules were shown to localize in tnt - like structures , but their intercellular transfer was not provenaccessed by the measurement of calcium fluxesproposed from the observation of a seamless transition between microtubules of the bridge with microtubular networks of both connected cellsnd : not determinedfig . 2transmission electron micrographs ( tem ) showing the ultra - structure of distinct tnt - like bridges in different cell types . ( a ) open - ended tnt connecting two pc12 cells reconstructed from images of two consecutive 80  nm sections . a continuous membrane is observed between the nanotube and the plasma membrane of the two connected cells . ( b ) close - ended tnt - like bridge connecting two t cells and displaying a junctional border , reconstructed from images of 13 consecutive 60  nm sections . the nanotube formed by one cell ( b2 ) protrudes into an invagination ( arrowhead ) of the connected cell ( b1 ) . scale bars , a , b , 1  m ; a1 , a2 , b1 , b2 , 500  nmfig . 3schematic representations of three distinct nanoscaled cellular protrusions and proposed modes of cell - to - cell communication . ( a ) a tnt - mediating membrane continuity between cells . ( a1 ) organelles like endocytic vesicles and mitochondria are transported uni - directionally between cells by an actin - dependent mechanism . ( b1 ) 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 . ( c ) cellular nanotube ( cytoneme ) extending toward a target cell by chemotaxis . ( c1 ) 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 ( a  c ) indicate the direction of transfer architecture of tnt between cultured pc12 cells . ( a ) 3d fluorescence image ( ( xy)-maximum projection of 40 consecutive 400  nm sections ) of a wheat germ agglutinin - stained tnt connecting two live pc12 cells . ( a1 ) ( xz)-projection in the plane of the tnt indicated in ( a ) . ( b ) scanning electron micrograph ( sem ) showing the ultra - structure of a tnt between two pc12 cells . scale bars , a , a1 , b , 5  m ; b1 , b2 , 500  nm tnt - like structures in vitro only those publications that fulfill at least one of the listed criteria are shown accessed by electron microscopy gurke , s. , barroso , j. , bukoreshtliev , n. , gerdes , h .- h . , unpublished data these molecules were shown to localize in tnt - like structures , but their intercellular transfer was not proven accessed by the measurement of calcium fluxes proposed from the observation of a seamless transition between microtubules of the bridge with microtubular networks of both connected cells transmission electron micrographs ( tem ) showing the ultra - structure of distinct tnt - like bridges in different cell types . ( a ) open - ended tnt connecting two pc12 cells reconstructed from images of two consecutive 80  nm sections . a continuous membrane is observed between the nanotube and the plasma membrane of the two connected cells . modified from rustom et al . ( b ) close - ended tnt - like bridge connecting two t cells and displaying a junctional border , reconstructed from images of 13 consecutive 60  nm sections . the nanotube formed by one cell ( b2 ) protrudes into an invagination ( arrowhead ) of the connected cell ( b1 ) . scale bars , a , b , 1  m ; a1 , a2 , b1 , b2 , 500  nm schematic representations of three distinct nanoscaled cellular protrusions and proposed modes of cell - to - cell communication . ( a ) a tnt - mediating membrane continuity between cells . ( a1 ) organelles like endocytic vesicles and mitochondria are transported uni - directionally between cells by an actin - dependent mechanism . ( b1 ) 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 . ( c ) ( c1 ) 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 ( a  c ) 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 ( for a comprehensive overview see ( gerdes et al . tnt - like bridges between dendritic cells ( dc ) ( watkins and salter 2005 ) and perhaps also those connecting neonatal rat cardiomyocytes ( cm ) and adult human endothelial progenitor cells ( epc ) ( koyanagi et  al . 2005 ) , appear to be most closely related to the tnt described for pc12 cells . the flow of cytoplasmic molecules suggests membrane continuity between connected cells ( table  1 ) . this contrasts the tnt - like bridges between t cells , which have been characterized lately in considerable detail ( sowinski et al . the observed t - cell nanotubes have an average length of 20  m , a diameter of 180380  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 ( fig . 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 ( fig . this was concluded from the observation that nanotubes between t - cells ( i ) do not facilitate the transfer of cytoplasmic molecules , ( ii ) do not permit free diffusion of fluorescent plasma membrane components and ( iii ) display a junctional border between the nanotube and the connected t - cell at the ultra - structural level ( fig . accordingly , they were classified as  not open - ended  cellular nanotubes ( fig . other identified close - ended nanotubular bridges that share some morphological features with tnt are murine leukemia virus - induced  filopodial bridges 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 ( 0.7  m diameter ) , which contained both f - actin and microtubules ( nfelt et  al . 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 ( nfelt et  al . thin , and thicker microtubule - containing connections were also found between prostate cancer cells ( vidulescu et  al . 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 ( ramrez - weber and kornberg 1999 ) . they are thought to extend toward the target cells by chemotaxis ( ramrez - weber and kornberg 1999 ) ( fig . 3c ) to accomplish the receptor - mediated uptake of the secreted morphogen decapentaphlegic ( dpp ) during spatial patterning ( hsiung et al . once endocytosed , the morphogen is thought to be delivered to the cell body by retrograde transport along the cytoneme ( fig . 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 ( bnl)-fibroblast growth factor ( fgf ) signaling , appear to do so ( sato and kornberg 2002 ) . the identification of tnt - like structures in cell cultures has been mainly based on the morphological criteria defined for pc12 cells ( rustom et al . nonetheless , heterogeneity regarding formation , structure , and functional properties across cell types and even within a given cell type has emerged ( table  1 ) . 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 . 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(s ) 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 ( rustom et  al . 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 ( ramrez - weber and kornberg 1999 ) ( fig . 3c ) or murine leukemia virus ( mlv)-induced nanotubular bridges emanating from non - infected fibroblast cells toward infected cells ( sherer et  al . the observation that tnt - like bridges also emerge when attached pc12 ( rustom et  al . 2008 ) depart from each other , led to the proposal of a second mechanism of tnt formation ( gerdes et  al . 2007 ; 2004 ) , 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 ( sowinski et al . 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 ( nfelt et al . actin polymerization is thought to be a key event for both mechanisms of tnt formation ( gerdes et  al . 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 ( rustom et al . 2004 ) , and tnt - like structures between t cells only form if the cells diverge with a speed below that of processes driven by actin polymerization ( cameron et al . ( 2005 ) 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 ( sowinski et al . 2008 ) , pc12 cells or normal rat kidney ( nrk ) cells ( bukoreshtliev , n. , gerdes , h .- h . , unpublished data ) 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 ( e.g. ruffles , invaginations ) , controlled by the cytoskeleton , to provide a buffer against membrane tension over several micrometers of tube elongation ( raucher and sheetz 1999 ; sun et  al . thus , the available membrane reservoir may restrict the number , total length , and lifetime of tnt - like connections for a given cell . 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 ( table  1 ) . 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 ( rustom et al . , unpublished data ) as well as mitochondria ( koyanagi et  al . 2005 ) were shown to traffic uni - directionally along tnt - like structures between cells over long distances ( fig . 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 , these organelles are one of the major reloading points for a variety of signaling complexes resulting from the endocytosis of activated cell surface receptors ( miaczynska et  al . once delivered to the target cells , the transferred endocytic vesicles are able to fuse with their counterparts ( rustom et  al . 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 ( koyanagi et  al . 2005 ) ( fig .  4 ) . 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 ( csords 2006 ; spees et  al . the obtained data are consistent with a tnt - related delivery , although alternative mechanisms were not excluded . 4emerging physiological implications of tnt - like structures emerging 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 ( gpi)-gfp ( nfelt and davis 2004 ; nfelt et  al . 2004 ) and the farnesylation signal of c - ha - ras fused to the c - terminus of egfp ( egfp - f ) ( rustom et  al . 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 ( dr . r. d. salter , univ . of pittsburgh , personal communication ) . it is of note that major histocompatibility complex ( mhc ) class i receptors were detected at the entire length of tnt - like bridges connecting immune cells ( nfelt et al . this points to a key role of tnt in a faster and more efficient presentation of antigens , in particular at the immunological synapse ( groothuis et  al . 2005 ; williams et  al . 2007 ) ( fig .  4 ) , defined as a highly specialized interface between immune cells ( norcross 1984 ; paul and seder 1994 ) . 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 ( stinchcombe et  al . 2001 ) . the characterized  membrane bridges  had a diameter of 5095  nm as observed by electron microscopy ( stinchcombe et  al . 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 ( rustom et al . 2004 ) , gfp between cm and epc ( koyanagi et al . 2005 ) , and the fluid phase marker lucifer yellow between thp-1 monocytes ( watkins and salter 2005 ) ( table  1 ) . 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 ( rustom et al . 2004 ) . thus , tnt - like structures , like pd , may have a variable size exclusion limit depending on the cell type ( gerdes et al . 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 ( watkins and salter 2005 ) ( table  1 ) . 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  m away from the point of stimulation . the elicited calcium wave propagated uni - directional with an initial speed of 35  m / s that slowed down rapidly to 1015  m / s ( watkins and salter 2005 ) . 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 ( table  1 ) . 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 ( watkins and salter 2005 ) . thus , this study provided the first evidence for functional connectivity accomplished by tnt and suggests a crucial role of these structures in immune defense ( fig . an emerging topic is the efficient spread of pathogens , like bacteria and mammalian viruses , along cell membrane protrusions ( figs .  3 , 4 ) . by using established or induced nanotubular connections between cells , pathogens circumvent the rate - limiting step of diffusion and efficiently accelerate their spreading ( sherer et  al . 2007 ; 2008 ) . among the viruses shown to exploit cellular protrusions to transfer from cell - to - cell are the vaccinia virus ( cudmore et  al . 2005 , 2006 ) , the herpes simplex virus ( la boissire et  al . 2004 ) , mlv ( sherer et  al . 2007 ) and human immunodeficiency virus type 1 ( hiv-1 ) ( sowinski et  al . we will focus on the latter two , which were characterized in much detail and gave rise to two different mechanisms of virus transmission . 2008 ) proposed membrane nanotubes between t cells as a new route for hiv-1 transmission ( fig . 3b1 ) , in addition to cellular contact points known as virological synapses ( jolly et  al . , they demonstrated its uni - directional move through innate nanotubular bridges and subsequent transmission into connected t cells ( sowinski et  al . 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 ( sherer et  al . 2007 ) . 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 ( sherer et  al . these filopodia are proposed to stably anchor to the cell body of an infected cell by viral envelope glycoprotein ( env)/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 ( sherer et  al . in addition to viruses , bacteria were reported to surf along tnt - like structures connecting macrophages ( nfelt et  al . 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 ( rustom et al . 2004 ) ( gurke , s. , gerdes , h .- h . , unpublished data ) as well as mitochondria ( koyanagi et  al . 2005 ) were shown to traffic uni - directionally along tnt - like structures between cells over long distances ( fig . 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 , these organelles are one of the major reloading points for a variety of signaling complexes resulting from the endocytosis of activated cell surface receptors ( miaczynska et  al . once delivered to the target cells , the transferred endocytic vesicles are able to fuse with their counterparts ( rustom et  al . 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 ( koyanagi et  al . 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 ( csords 2006 ; spees et  al . the obtained data are consistent with a tnt - related delivery , although alternative mechanisms were not excluded . 4emerging physiological implications of tnt - like structures emerging 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 ( gpi)-gfp ( nfelt and davis 2004 ; nfelt et  al . 2004 ) and the farnesylation signal of c - ha - ras fused to the c - terminus of egfp ( egfp - f ) ( rustom et  al . 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 ( dr . r. d. salter , univ . of pittsburgh , personal communication ) . it is of note that major histocompatibility complex ( mhc ) class i receptors were detected at the entire length of tnt - like bridges connecting immune cells ( nfelt et al . this points to a key role of tnt in a faster and more efficient presentation of antigens , in particular at the immunological synapse ( groothuis et  al . 2005 ; williams et  al . 2007 ) ( fig .  4 ) , defined as a highly specialized interface between immune cells ( norcross 1984 ; paul and seder 1994 ) . 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 ( stinchcombe et  al . had a diameter of 5095  nm as observed by electron microscopy ( stinchcombe et  al . 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 ( rustom et al . 2004 ) , gfp between cm and epc ( koyanagi et al . 2005 ) , and the fluid phase marker lucifer yellow between thp-1 monocytes ( watkins and salter 2005 ) ( table  1 ) . 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 ( rustom et al . 2004 ) . thus , tnt - like structures , like pd , may have a variable size exclusion limit depending on the cell type ( gerdes et al . 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 ( watkins and salter 2005 ) ( table  1 ) . 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  m away from the point of stimulation . the elicited calcium wave propagated uni - directional with an initial speed of 35  m / s that slowed down rapidly to 1015  m / s ( watkins and salter 2005 ) . 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 ( table  1 ) . 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 ( watkins and salter 2005 ) . thus , this study provided the first evidence for functional connectivity accomplished by tnt and suggests a crucial role of these structures in immune defense ( fig . an emerging topic is the efficient spread of pathogens , like bacteria and mammalian viruses , along cell membrane protrusions ( figs .  3 , 4 ) . by using established or induced nanotubular connections between cells , pathogens circumvent the rate - limiting step of diffusion and efficiently accelerate their spreading ( sherer et  al . 2007 ; 2008 ) . among the viruses shown to exploit cellular protrusions to transfer from cell - to - cell are the vaccinia virus ( cudmore et  al . 2005 , 2006 ) , the herpes simplex virus ( la boissire et  al . 2004 ) , mlv ( sherer et  al . 2007 ) and human immunodeficiency virus type 1 ( hiv-1 ) ( sowinski et  al . 2008 ) . 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 . 2008 ) proposed membrane nanotubes between t cells as a new route for hiv-1 transmission ( fig . 3b1 ) , in addition to cellular contact points known as virological synapses ( jolly et  al . 2004 ; jolly and sattentau 2004 ; sol - foulon et  al . 2007 ) . 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 ( sowinski et  al . 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 ( sherer et  al . 2007 ) . 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 ( sherer et  al . these filopodia are proposed to stably anchor to the cell body of an infected cell by viral envelope glycoprotein ( env)/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 ( sherer et  al . in addition to viruses , bacteria were reported to surf along tnt - like structures connecting macrophages ( nfelt et  al . 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 ( fig . 3 ) , with the exception of a microtubule - dependent , bi - directional movement of organelles in thick bridges connecting macrophages ( nfelt et  al . 3a1 ) due to the presence of the barbed - end directed actin - specific motor myosin va in tnt ( rustom et  al . 2004 ; zhu et  al . 2005 ) ( bukoreshtliev , n. , gerdes , h .- h . , unpublished data ) ( table  1 ) partially co - localizing with the respective organelles ( rustom et  al . this model is supported by the low transport velocity in the range of actin - dependent transport ( rustom et  al . 2004 ) 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 ( sowinski et al . 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 ( sherer et  al . 2007 ) . 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 ( fig . 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 ( rustom et  al . , gating mechanisms may exist to control this open - ended transport ( gerdes et  al . 2007 ) . 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 ( sowinski et  al . 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 ( reviewed in gerdes et  al . 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 ( rrth 2003 ) ( table  2 ) 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 [ m ] diameter [ nm ] growth speed [ m / min ] thin , elongated , active filopodia and lamellipodia primary mesenchyme sea urchin embryos ( gustafson and wolpert 1967 ; miller et al . 1995 ) f - actinnd 8020040025cytonemes drosophila wing imaginal discs ( ramrez - weber and kornberg 1999 ) f - actin nd < 700 200 15 cytoneme - like mouse limb bud cells ( ramrez - weber and kornberg 1999 ) ; drosophila wing imaginal discs ( jacinto et al . 2000 ) f - actinnd 10nd 1apical peripodial extensions drosophila wing / eye imaginal discs ( gibson and schubiger 2000 ) microtubules mitochondria530 nd nd myopodia drosophila embryonic muscle cells ( ritzenthaler et al . 2000 ; ritzenthaler and chiba 2003 ) f - actin nd 40 nd nd myopodia - like mouse transverse muscle of thorax ( misgeld et al . 2002 ) ; xenopus ( kullberg et al . 1977 ) nd nd 10nd nd cellular extensions drosophila eye imaginal discs ( chou and chien 2002 ) f - actin scabrous vesicles 40 nd nd apical and lateral cell protrusion drosophila wing imaginal discs ( demontis and dahmann 2007 ) f - actin gfp - rab5 , gfp - tkv110 200 nd data from miller et al . ( 2000)these molecules were shown to localize in cell protrusions , but their intercellular transfer was not provendata from misgeld et al . ( 2002)nd : not determined selected examples of cell protrusions in tissue data from miller et al . ( 1995 ) data from jacinto et al . ( 2000 ) these molecules were shown to localize in cell protrusions , but their intercellular transfer was not proven data from misgeld et al . ( 2002 ) the formation of cellular extensions (  pseudopods  ) during embryogenesis became first evident by studying sea urchin morphogenesis ( gustafson and wolpert 1967 ; miller et  al . 1995 ) , 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 ( ramrez - weber and kornberg 1999 ) . this discovery inspired further studies describing other cellular protrusions in the wing disc extending not only within ( chou and chien 2002 ; demontis and dahmann 2007 ) but also between cell layers ( demontis and dahmann 2007 ; gibson and schubiger 2000 ) . in a very recent study on live wing discs , demontis and dahmann ( 2007 ) 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 ( demontis and dahmann 2007 ) . thin , actin - based membrane extensions were also found to be crucial for embryonic dorsal closure in drosophila ( jacinto et  al . 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 ( bard 1992 ) , and wound healing ( wood et  al . 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  m 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 ( ritzenthaler et  al . finally , long , thin cellular extensions are also associated with guided cellular migration during development ( ribeiro et  al . 2002 ; sato and kornberg 2002 ) , immune defense such as the migration of macrophages through the endothelial cell layer ( blue and weiss 1981 ) and tumor cell invasion ( vignjevic and montagnac 2008 ) . certainly , there are many more examples of long cellular nanotubes in tissue involved in cellular communication besides the ones listed here ( table  2 ) . 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 .
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 ( tnt ) , 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 .