Cellular communication is an old story. Multiple mechanisms for communication, including signal transduction, material transportation supported by multiple sub-cellular structures such as synaptic vesicle and junction complex, have remained to be 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 the 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 Materials

Cell preparation

We tried three approaches to induce the formation of nanotubes among Hela cells: Cholera toxin B induction, M-sec transfection and harsh environment induction (low pH, high glucose and low serum concentration), with the last one of highest efficiency. Hela cells with induced nanotubes and E.coli marked by Green Fluorescence Protein (GFP) were prepared for electroporation. After replacing culture medium with PBS, Hela cells were incubated with E.coli during exponential phase for 30 minutes. Before electroporation, the PBS was removed as thoroughly as possible in case of short circuit.

Electroporation

Illustrated schematically in Figure 1. After PBS was removed, two electrodes were inserted into the culture dish with their tips directly attached to the cell layer. Electrical pulses were delivered from a stimulator to a regular electric isolator, which was drived to output weak positive current (27–40 μA). The current flowed through the Hela cells from one electrode to the other. The waveform (square wave, 5Hz, 25ms) and amplitude of injected current were monitored via an oscilloscope to measure the voltage drop across a series resistor (75 KΩ), which was 2–3V.

Dye loading and two-photon Ca2+ imaging

We loaded nanotube-linked Hela cells with a Ca2+-sensitive dye and observed the Ca2+ flow through two-photon microscope. A total of 1μL Fluo-2 was diluted 1:1000 with 1× PBS. A culture dish of Hela cells was incubated with this solution for 35 minutes at 37℃ to full loading. After confirming loading, the Fluo-2 dye was completely washed off by PBS, repeating 3 to 4 times. Two-photon imaging of changes in Ca2+ fluorescence in Hela cells was monitered with a costum-built microscope coupled with a Mai Tai mode-locked Ti:sapphire laser (730–740 nm).

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 were then able to observe some of the inter-cellular communications that had never been documented in this cell line, including the complete exchange of organelles and cell-to-cell Ca2+ flow. But communication is merely 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 have learned during the experiments, we have envisioned the following prospectives in further nanotubule researches and applications:

1. From single cell to syncytium, finally to multicellular organism？

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

2. An approach to establish another version of a nervous system?

Some of the latest outcomes of the harsh environment simulation induction were absolutely sensational, with some members of the syncytial entity having developed the similar structure to neurons, connecting multiple parts of the entity via both types of nanotubes. Since Ca2+ flow can be passed on to other cells via nanotubes, a process that may effectively alter the inner bio-chemical activities of the downstream cells, nanotubes 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 nervous tissue.

3.Potential application in curing cancer?

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

Electroporation part

1. Protein that enables synchronization within bacteria colony and facilitating bacterial distribution.

2. Endosymbiosis

It can be considered as a bonus that with the successful introduction of bacteria into eukaryotic cells via modified electroporation, we actually become the very first ones to acquire a direct evidence in support of the endosymbiosis hypothesis. Electric shock and little conductive solution are two essential elements ensuring bacterial entrance, which can simulate an environmental condition that could be easily obtained back in ancient times. Inspired by the experimental results, we are so astonished to realize that the process of establishing endosymbiosis might happen much more frequently than we used to think it would. Given a weak positive current, bacteria can be driven to surround the eukaryotic cells and then enter the plasma via the instantaneous pores on the cell membranes.