Table 1.

Open questions in the area of TNT and gap junctions.

Theme	Specific questions	Workshop output
Pathophysiological function of TNTs	Why are TNTs induced in disease?	Hijacking development and/or an evolutionary response Stress induction Specificity in cargo delivery—energy conservation Exchange of genetic material to support disease or rescue damage cells from cell death
Translational relevance of TNTs in disease	TNTs are thought to play a role in disease—which disease(s)?	Diseases include cancer, neurodegenerative disorders, tau related diseases, HIV, lysosomal disorders, inflammation, parasitic infections (Malaria)
	What role do TNTs play is disease?	Promoting the disease (e.g., spread of virus, protein aggregates), mitochondria between cancer cells (chemo-resistance) Rescuing the cell function (e.g., lysosomal and mitochondrial transfer to defective cells)
	Elucidating Normal Physiologic functions of TNTs	Facilitating cell contact during development (e.g., cytonemes) Promoting cell communication (e.g., Signaling) between distant cells Immune response and organelle exchange mechanisms Function in stem cell biology and tissue repair Diverse heterogeneity of TNTs (phenotype/functions/disease/health) Proven importance/roles of TNTs for immune response Multi-functional cargo (“FedEx”-like)
	What are the key learnings	Cell structure is important for spread/progression of the disease by transferring, e.g., infectious agents between cells and for cell-to-cell communication, e.g., during development, tissue regeneration Potential therapeutic target to block disease progression TNT formation during development (e.g., CNS), pathological events (pathogens, tumor cells, misfolded/aggregated/stress protein), during regeneration process (stroke), in inflammation/immune response and drug delivery Importance of identifying mechanism of actin/motors that drive TNTs Elevating research beyond in vitro, in vivo and 3D studies Importance of examining heterotypic TNT interactions, e.g., cancer-to-stem cells, cancer/stroma To examine the immense heterogeneity of definition of TNTs
Cellular mechanisms of TNTs	What is known about TNT cell biology?	Strong evidence of TNT formation in vitro (e.g., infectious disease, oncology, neurology, development) Intercellular communication /signaling/cargo/dyes Inducible (infection/inflammation) A Large variety of cells capable of TNT formation Some evidence of TNT formation in vivo (oncology) Evidence that M-sec, myosins, F-actin, and calcium transfer are involved Shaking/physical disruption blocks TNT formation Gap junctions may play a role in TNT connecting to receiving cells (Focus if this review)
	What is the overlap, and what are the potential differences, of TNT biology in normal cells vs. in disease, and between different diseases?	Differences—induction of TNT seems to be associated with “diseased” cells The direction of cargo communication Cell types/microenvironment (tumor/inflammation) In diseased cells, F-actin polymerization is increased.
	How does a donor cell “decide” what organelles, molecules or signals transfer through TNTs?	Key factors include: ◦ Variety of TNTs ◦ Different triggering factors (pathogen, metabolic stress, e.g., reactive oxygen species) ◦ Selectivity of organelles and direction of travel ◦ Uni-/bi-directional depends on cell type &/or cargo Stress response Preferential transfer of mitochondria Virus hijack TNTs
	TNT research has advanced over the last 10 years—what are the key focus areas to advance this science?	Mechanism of TNT formation—trigger, direction, cargo/content, structure, response Better characterization—different types of TNTs, types of cells able to make TNTs In vivo evidence of TNT—in development, in disease model, regeneration mechanism (stem cells) Develop/test TNT blockade strategies and TNT induction mechanism Delineate relationship between TNTs and inflammation/immune response stromal The following items are needed: ◦ Chemical tools ◦ Common mechanism of transfer ◦ Selectivity ◦ A TNT biomarker ◦ In vivo evidence Cargo identification Regulation and induction/suppression Accept TNT heterogeneity (no simple narrow definition to make this science grow) Technology hurdles: Need higher resolution microscopy (e.g., EM, cryoEM, organelle level resolution, identifying cellular structure “signatures”) Collaboration with medicinal chemists to synthesize inhibitors of key TNT-drivers (e.g., M-sec) Proteomics to identify TNTs and their contents Targeted drug delivery via TNTs (e.g., siRNA) “How does the TNT know where to go?” cell sensing mechanisms? Translational relevance: identify strategic approaches that are disease specific Standardization of terminology Broader definition, including subtype descriptors In vivo data, especially patient data Better, specific markers → enable 3D culture experiments, in vivo, etc TNT biochemistry—reconstitute in a cell-free system