Cellular communication is an old story. Multiple communication mechanisms, including signal transduction, material transportation supported by multiple sub-cellular structures such as synaptic vesicle and junction complex, remain the hot spots of cell biology for many years. However, in addition to all the above-mentioned, are there any other communication mechanisms more intimate and straightforward? Take a look at the plasmodesma, which is a special cellular structure connecting neighboring plant cells. The two cells on each end of the plasmodesma share a certain amount of cytoplasm. Therefore, it serves as an important and unique pathway of communication in the plant kingdom. So is it possible for animal cells, which lack the rigid structure of cell wall, to form and maintain such a plasmodesma-like structure? May it succeed, what changes can be observed among the altered cells with this new type of communication? These are the questions that we tried to answer as we started out this project.

Introduction

The second project of Team Fudan-Lux is about constructing a brand-new biological model using a recently discovered cellular structure termed Tunneling Nanotubes(TNT) and bacteria containing the green fluorescence protein. By inducing and stabilizing TNTs between certain types of malignant tumor cells, a cellular network could be obtained. Then the bacteria containing GFP is introduced into the tumor cells by microinjection. By doing so, a new type of biological system is created. More importantly, what we want to study here, is the behavior of the injected bacteria within the tumor cells. Since TNTs formed between cells act as super highways for material transportation, bacteria thus can move from one cell to another via TNTs. Given the condition that bacteria would tend to choose the most suitable place for them to live in, in the least energy-consuming way, a distribution pattern thus can be obtained which have the characteristic of the least increase of entropy. By building such a model, we want to simulate certain types of problems in the real life that can’t be solved by simple computation, e.g. traffic jams between cities, and provide solutions to them.

Methods and Material

The cell line that we chose here was Hela (need that specific information!!!), given its property of easy culture, proper life span and replication cycle, and ability to withstand a certain degree of harsh environment. Nanotube induction M-sec CTB harsh-environmental simulation induction immuno-fluorescence Cytoskeleton supporting nanotubes were confirmed by immuno-stainning. Phalloidin Verification of cellular communication via nanotubes mitochondria stainning Ca2+ flow recording harsh environment recovery and further culture and observation Electroporation

Results

Nanotube induction (three pictures representing the different induction conditions & one showing the normal hela cells) As it can be seen in these pictures, hela cells under induction would form a significantly higher amount of nanotubes in comparison with normal hela cells. Cells underwent harsh-environmental simulation induction displayed the most prompt and radical cellular structure changes. Two types of nanotubes have been observed: one was a wide (with a proximate diameter of ___) cell protrusion-like structure that reached out from one cell and directly touched another cell even across a rather long distance; another type of nanotube was comparatively much thiner, but in a rather great number. The latter type of nanotubes could be found at the end of the former ones, thereby further connecting distant cells; or right from the middle of the cell bodies linking several neighboring cells all at once. Both types were supported by F-actin and were non-adherent to the substratum. Cultured cells stained with mitotracker after nanotubule induction and stabilization were then placed under microscope for observation. As it can been seen in this figure, mitochondria could travel from one cell to another via nanotube, demonstrating the property of material transportation of these induced nanotubes. Moreover, Ca2+ dye loading and two-photon Ca2+ imaging further confirmed the communication via Ca2+ flow between two connected cells. With these experimental results available, we can basically confirm the cellular structures that we induced and stabilized here are those we anticipated. When the harsh-environmental simulation induction prolonged, the cells with nanotubes underwent more radical changes of cellular structures. After 5 days’ induction, most living cells tended to distribute most of their plasma into wide and elongated nanotubes, resulting in an octopus-like shape of each single cell and a web-like system among the whole cell colony. Incubating these cells with normal culture media for one to two days, several originally distant cells connected merely by nanotubes moved towards each other and finally clustered together, forming one big syncytium that had interchangeable plasma and organelles via nanotubes. As cells of this syncytium divided, its state remained as the newborns, with nanotube-linked neighboring cells as well. Further Ca2+ dye loading and two-photon Ca2+ imaging demonstrated a synchronization of Ca2+ flow among each individual within this syncytium. Electroporation& Microscopic imaging Cultured Hela cells after electroporation were then placed under differential interference contrast(DIC) microscope for bacterial entrance verification. Wild type E.coli MG1655 expressing GFP could be seen inside the hela cells clearly. Most entered bacteria tended to cluster around the nucleus, a phenomena we reasoned that could optimize the bacterial distribution into daughter hela cells. Also, we observed a significantly higher GFP intensity given by the bacteria within hela cells, compared to bacteria stayed on the outside, which indicate that the plasma may be a more favorable place for this facultative anaerobe to live in. After executed harsh-environmental simulation induction among bacteria-breeding hela cells, symcytia similar to the former ones were obtained, with a typical spider web outlook. Visible E.coli MG1655 could be seen traveling from one hela cell to another via nanotubes, indicating a non-seletivity among the transporting cargo of these nanotubule highways. However, due to the viscosity of plasma and the retardance of the cytoskeleton, E.coli MG1655 within hela cells did not exhibit the mobility that we desired. In addition, the relationship between E.coli MG1655 and hela cells appeared to be rather intense as usually only one of these two parties can survive after a few days of incubation of this temperal man-made endosymbiotic system.

Future prospective

Nanotube part

In this project, we successfully introduced a brand-new type of communication mechanism into cultured hela cells. As the first ones to establish such cellular structure in hela cells, we then were able to observe some of the inter-cellular communication that had never been documented in this cell line, including the complete exchange of organelles and cell-to-cell Ca2+ flow. But communication is just the first step. Changes made possible by such communication, whether between or within cells, we believe, are of even greater significance. So far we have already observed obvious cellular behavioral changes------single cells forming nanotubes in an attempt to reach out and touch some other cells. Based on what we’ve learned during the experiments, we have envisioned the following prospectives in further nanotubule research and application:

1. From single cells to syncytium to multicellular organism？

Combining the conditions under which we conducted the recovery experiment with the later visible cellular structral changes, we further came up with a thought of how such structure and communication mechanism functioned to boost the evolution from single cell to multi-cellular organisms. As we have already observed how the construction of the nanotubule network affect the organization among cell colonies------from single cells to a syncytial entity, which then move on to a multicellular colony among which a Ca2+ flow synchronization could be obtained.

2. A way to establish another version of a nerve system?

Some of the latest outcome of the harsh environment simulation induction were absolutely sensational, with some member of the syncytial entity developed the similar structure of neurons, connecting multiple parts of the entity via both types of nanotubes. Since calcium ion flow could be passed on to other cells via nanotubes, a process that may effectively alter the inner bio-chemical activities of the downstream cells, nanotubes thus could be considered as a counterpart of the axon of a neuron. With further modified induction solutions, we might be able to establish a multicellular structure that has certain features of the nerve tissue.

3.Potential application in curing cancer?

As a communication channel which can facilitate the transportation of certain cellular contents, cells with large amount of nanotubes thus may also perform as a drug delivering system. By establishing cell lines that possess the property of high nanotubule formation and introducing them into tumor tissues, modified cells can form multiple connections with native cancer cells. If we introduce foreign genes encode for elements that might disturb the inner machinery of cancer cells, which could be produced by the modified cells and transported to the cancer cells via newly formed nanotubes. Such a new type of drug delivering system can thus achieve direct drug-delivery which could overcome some of the major drawbacks of present therapy for curing cancer.

Electroporation part

1. Protein that enables synchronization among bacterial colony and facilitating bacterial distribution.

2. Endosymbiosis

It could be considered as a bonus that with the successful introduction of bacteria into eukaryotic cells using modified electroporation, we actually became the very first ones to provide with a direct evidence in supporting the endosymbiosis hypothesis. Electrol shock and little eletro-conductive solution are two essential aspects ensuring bacterial entrance, an environmental condition that could be easily obtained back in those ancient days. Inspired by the experimental results, we were so shocked to realize that the process of establishing endosymbiosis might happen much more frequently than we once thought it would. Given a little charge of electricity and a conductive environment, bacteria move closely around eukaryotic cells could then enter the plasma.