the property that most clearly separates tnts from other cellular protrusions in vitro is their straight , bridge - like structure , interconnecting cell pairs . in vitro imaging has shown that the length of tnts displays large variation , differing between cell lines ( table 1 ) . tnts connecting t cells , for example , were reported to have an average length of 22 m , whereas in pc12 cells , the length was found to be much less . the tnt lengths can vary as the connected cells migrate and the distances between them change , indicating that tnt length can be dynamically regulated . in addition , some cells show a negative correlation between the tnt lifetime and the cell migration speed . statistical analysis of tnt length will provide information about the effective distance for tnt formation , and also the threshold distance for tnt - dependent cell - to - cell communication . wga , wheat germ agglutinin ; did , vybrant did celllabeling solution ; sem , scanning electron microscopy ; tem , transmission electron microscopy ; dic , differential interference contrast ; afm , atomic force microscopy . * unpublished data ; * * all nanotubes contained actin , and a subgroup also contained microtubules ; * * * all nanotubes contained microtubules , and a subgroup also contained actin ( 65% ) . measuring the diameter of tnts using light microscopy can not be done with adequate accuracy due to the resolution limit . transmission electron microscopy analysis has revealed that tnts have a diameter in the range of 50200 nm in pc12 cells and 180380 nm in t cells ( table 1 ) . however , to preserve and search for intact tnts in series of sample slices is laborious . confocal microscopy has shown that some tnts reach thicknesses of over 700 nm , which could be due to incorporation of additional components inside the tnts , such as microtubules . it should also be noted that multiple thin tnts could stick together to form what looks like a single , thick tnt ( unpublished data ) . since cells after division sometimes form transient thin intercellular connections containing a midbody ring , a double labeling can help to distinguish tnts from incompletely divided cells . tnts are not empty membrane tubes , but filled with cytoskeletal filaments ( table 1 ) . f - actin is found in most tnts , spanning uniformly along their entire length , and is thus an important labeling target in tnt - imaging . f - actin also plays a crucial role in the formation of tnts , as shown in experiments where incubation with f - actin depolymerization drugs , such as cytochalasin b , inhibit tnt formation . in addition , evidence show that various cellular components are transported inside tnts in the speed range of f - actin - associated myosin - motors . besides f - actin , microtubules are also detected in tnts in a few cell lines , such as immune cells , between primary neurons and astrocytes , and in huvec cells during cancer - induced angiogenesis . why and how microtubules are present in some tnts remains to be investigated . as with f - actin and myosin , microtubules could serve as tracks for transport of cargo via a kinesin / dynein - mechanism . furthermore , microtubule - filaments have shown a bending stiffness many orders of magnitude higher than that of actin - filaments . thus , incorporation of microtubules could provide a high degree of rigidity and longer lifetime to the tnt . transmembrane proteins and membrane - binding proteins , such as n - cadherin and myosin x , are considered necessary in the recognition of and attachment to target cells during tnt formation ( unpublished data ) . for example , interposed gap junctions on the tnt / cell - contact site allow transmission of depolarization signals . moreover , the accumulation of mhc class i chain - related protein a ( mica ) at the tip of nanotubes of natural killer cells can induce immune responses in target cells . in t cells , the transfer of endogenous fasl from effector cells can result in apoptosis in the receiving cell . finally , certain membrane components could accumulate passively along tnts , thus becoming a potential marker for the imaging of tnt - like structures . in support of this theory , experiments with liposomes have shown that both specific proteins and lipids can be sorted spontaneously into artificial nanotubes . in addition to the tnts described above , some extracellular protrusions can be considered  tnt - like ,  as they share some of the characteristics of tnts ( table 2 ) . besides filopodia cytonemes are long and thin protrusions containing f - actin found in the drosophila imaginal disc . they facilitate peripheral uptake and subsequent transport of extracellular signaling molecules toward the cell body . it is not clear if these protrusions attach to other cells like tnts do , or if they just act as periscope - like sensors . another tnt - like structure was observed in cultured b cells upon antibody - opsinization . the streamers also contain f - actin , and do not form when incubated with the f - actin depolymerization drug cytochalasin d. however , the streamers , like cytonemes , do not necessarily attach to other cells . when they do make contact to other cells , evidence suggests that this confers a protective effect to complement - mediated cell lysis . in 2011 , a new kind of thin extracellular protrusions , called a nanopodium , was reported . nanopodia emanate from endothelial cells expressing the tetraspanin - like protein tm4sf1 , which is also found in puncta along the protrusions . being relatively deficient in f - actin , and also not necessarily attached to other cells , their function in intercellular communication is uncertain . although the morphology of the structures discussed above resembles tnts closely , they usually do not interconnect cells . however , it is reasonable to believe that the cell employs much of the same machinery to form these structures , and some could possibly be tnt - precursors under certain conditions since tnts can derive from filopodia . the search for tnts in tissue is important not only to establish their presence in multicellular organisms , but also as a key step to understand their physiological functions . the development of fluorescent protein tags and advanced confocal microscopy have facilitated tnt identification in vivo by enabling the labeling of specific proteins only present in a certain subpopulation of cells . in chick embryos , tnt - like structures were successfully identified between neural crest cells after this specific cell population had been labeled with fluorescent fusion proteins . there are similar findings from gastrulation - stage embryos , and in immune cells in the adult mouse cornea . tnts between neural crest cells have been shown to mediate the transfer of cellular material , indicating that tnts play a functional role also in vivo . to date , most of the reports on tnts in vivo are studies on embryos , which might hint at a role of tnts during the development of multicellular organisms . although the tnts in tissue share many morphological features with tnts in vitro , there are still dissimilarities that can be ascribed to the complex microenvironment in vivo . contorted tnts have been found both in healthy tissue , such as in the mouse cornea , as well as in tumor tissue . this effect could possibly be due to obstacles , such as other cells and dense extracellular matrix , preventing the protrusion from connecting at the shortest distance between the two cells . indeed , when there are no obstacles between the cells , such as between the rims of the neural folds during neural tube closure , straight tnt - like structures can be found in vivo . in addition , extracellular matrix could in principle provide structural stability and protection from external forces when it is surrounding the tnts . in a recent paper elaborating on tnts in the cornea , the lifetime was measured to be more than 90 min . a study on primary t cells inside an artificial 3d matrix revealed long - lived tnts with curved morphologies , in accordance with in vivo studies . due to the difficulty of imaging tnts in vivo , details about the composition and structure of tnts in complex tissue environments remain to be investigated . certainly , such research will expand our knowledge about tnts , and may also give information about their function and the mechanism of formation in tissue . the heterogeneous morphology and composition of tnts suggests that tnts may form in different ways . according to live imaging of cultured cells , the formation of tnts occurs by filopodial interplay (  making contact  ) or cell dislodgement (  keeping contact  ) . recent research on the molecular level has revealed that the unconventional motor protein myosin x , usually associated with filopodia , promotes tnt formation by interacting with several transmembrane proteins , supporting the notion that tnts can derive from filopodia ( unpublished data ) . moreover , the m - sec protein has been demonstrated to be an important regulator of tnt formation . it was also involved in p53 and mhc class iii protein lst1 induced tnt formation . however , recent observation that p53 is not a master protein for tnt formation in every cell type , and the fact that tnts have been observed between cells that do not express m - sec , suggest that different mechanisms of tnt formation may exist in different cell types . therefore , there may be a certain level of one - to - one relationship between the diversity of tnts and the molecular determinants of their formation . several studies support the notion of a correlation between the structure and the function of tnts . an interesting example is the tnt - dependent propagation of calcium - fluxes between cells , for which at least three ways have been described in different cell lines . first , tnt - mediated electrical coupling can transfer depolarization from one cell to another via interposed gap junctions , and thus elicit a depolarization - dependent calcium uptake in the connected cell through voltage - gate channels . second , ins(1,4,5)p3 receptors bound to the endoplasmic reticulum inside tnts actively propagate intercellular calcium signals along tnts via calcium - induced calcium release . lastly , calcium itself can diffuse directly through tnts , thereby rising calcium level in the receiving cell . obviously , in mediating this calcium flux , the tnts need to fulfill certain structural requirements . in the first model , the presence of gap junctions on the contact site between the tnt and the target cell is necessary to transfer electrical depolarization . in the two latter models , a tnt with a larger luminal diameter is required to allow the entry of endoplasmic reticulum or efficient diffusion of calcium from one cell to another . in addition , motor proteins might be necessary to mediate the entry of endoplasmic reticulum into tnts . thus , the structural and compositional properties of the tnts determine the possibilities and limitations of their functions that can be performed . future research on tnts will inevitably increase our knowledge about the morphological and structural diversity of tnts during embryo development , tissue homeostasis , and pathological processes . however , to resolve the molecular mechanisms underlying the diversity , as well as to depict the genetic , cellular and species - specific variation , represents a great challenge . in addition , to determine the relationship between the diversity and the different functions of the tnts , particular at the tissue level , is an important task in this research field .
tunneling nanotubes ( tnts ) are recently discovered thin membranous tubes that interconnect cells . during the last decade 
 , research has shown tnts to be diverse in morphology and composition , varying between and within cell systems . 
 in addition , the discovery of tnt - like extracellular protrusions , as well as observations of tnts in vivo , has further enriched our knowledge on the diversity of tnt - like structures . 
 considering the complex molecular mechanisms underlying the formation of tnts , as well as their different functions in intercellular communication , it is important to decipher how heterogeneity of tnts is established , and to address what roles the compositional elements have in the execution of various functions . here , we review the current knowledge on the morphological and structural diversity of tnts , and address the relation between the formation , the structure , and the function of tnts .