Hi Anh, this an interesting observation. There are some papers that describe gelatin fragments as being chemoattractants.

The cells you are working with must be polarized with a leading edge.

I would recommend adding dye quenched gelatin in your assay.

https://www.thermofisher.com/order/catalog/product/D12054

This gelatin is non-fluorescent until it is cleaved, releasing fluorescent gelatin fragments. This way you can follow their movements in 2D and 3D.

If your cells expressing the mutated protein show a non-distinct trail of fluorescence, it might mean that they have lost their polarity. A directional migrating cell would show a distinct zone of fluorescence at the leading edge.

A cell that migrates at random would not show a directional zone of lysis.

Hi Steingrimur,

The surprising bit is that actually the cells with the mutated protein show very very polarised end. So they have very broad lamellipodia at the front end and the cells glide very easily on top of the substratum, but their motion is directionless. What I mean is that they move in circular motion, like spinning around one point or going in circle. While the one with the normal WT protein, they almost like crawling instead of gliding. But they crawl in a very directional way, so they less frequently changing their direction but rather keep going forwards. They have protrusion rather than lamellipodia. 

In term of speed, the two have very similar speed. But in term of directionality, the mutated one doesnt seem to have that. In term of polarity, the mutated one have very very prominent lamellipodia at the leading edge.

So this is very difficult because we always thought that this mutant protein would make the cell more aggressive and metastasize and invade better. But how a circular motion make it any advantageous? 

Again, 3D invasion assay does show that these cells invade more. 

But if just base on 2D random migration assay, you wouldn't expect that at all. 

What do you think?

Hi Anh, as far as I know there are 2 major factors governing cells to migrate trough a matrix (gelatin or otherwise)

1) Proteinases. Many matrix proteins have cryptic RGD cell adhesion sites that are only exposed when cleaved. But this is gelatin. All of the RGD adhesion sites are already exposed.

2) Integrins. The avb3 and avb5 integrins are highly expressed on motile cancer cells, but since there are exposed RGD binding sites are everywhere in a gelatin matrix, they should not have a problem finding adhesion sites.  

I am a bit stumped, but it could be that the mutated cells need a signal for them to be directional. They may be idling racecars waiting for a signal.

Have you tried putting a blob of serum in gelatin on one side of the dish and see if that changes the behavior of the mutated cells? If they stop whirling around and home in on the serum blob, then they might just have the signal needed for directional migration. 

Hi Steingrimur,

Indeed. The phenotype confuses me quite a lot. These are transformed melanocytes too. So they contain the constitutively active mutant NRas which normally found in melanoma. 

The mutated cells glide really smoothly on the gelatin matrix with very beautiful lamellipodia, very persistent lamellipodia. But very very often they make a steer motion, and turning and start going in circle. But this is 2D matrix, so I appreciate this is different from 3D or physiological environments. But this is very difficult to explain. 

Hi Anh, as you probably know, melanomas are one of the nastiest and most metastatic cells out there.

Have you tried encasing them in gelatin with added DQ gelatin?

Melanomas are used to being in air-skin interfaces. That's where they develop and are detected.

Its what they do after they migrate into tissues is where they do the most damage.

Plus I am interested in their color. Are they like the mouse M16 melanoma that are black? Any changes in tyrosinase activities or melanin deposits.?

(BTW, if you recommend my answer, RG will forward that to my inbox. Otherwise I will have to look for your answer. Take care. Stenni) 

My cells still retain their normal colours, or at least between the mutated and the control cell line, they have similar colour, nothing different about them. 

But thank you very much for the help. I think its very unusual that these cells have such phenotype. Its still a mystery how this is relevant. 

Can you fix them and look closely, there must be some melanin deposits.

As for the relevance of the mutations, I think you have to go in vivo (if you have an animal facility).

Inject ~1000 cells of the wt and the mutant cells into the backs of SCID mice. To follow them you can pre label them with CellTracker green (from IVGN)

The dye does not harm the cells and you can follow their progeny to where they migrate.

Article A cell transportation solution that preserves live circulati...

Hi Stenni,

Oh right, that does sound reasonable to me. I think intra vital imaging is a good idea. We are planning on that too. The reason I ask is because 3D experiment definitely show a stronger invasion with cell containing the mutation compared to the wild type. But on 2D substrate, the circular movement of the cell doesnt seem to match up with the deep invasion in 3D, or maybe it does but we just dont know the mechanism behind. 

Hi Anh, in vitro experiments can only yield limited data based on the their design.

The circular movement of your cell mutants in 2D could be an indicator that they are actively looking for a chemotactic signal. If the gelatin matrix does not have a directional chemotactic signal, they might not move in a direct pattern.

Your WT cells might be migrating because they are primed to secrete proteinases and move into zones of lysis. 

Only in vivo experiments might solve this issue, but before that,  please include a blob of FBS in gelatin on one end of the plate and see if that induces directionality of the mutant cells.   

 Hi Stenni,

I think that's a reasonable approach too. I did talk with my colleagues and a chemotaxis assay may explain this.  

Thank you very much for discussing with me. Sometimes I just feel like all these data that I got dont make any sense at all and that just makes me nervous and scared. But at the same time, such prominent and unexpected phenotype like this may hint something we dont know about the cell. 

Hi Anh, being scared, nervous and doubting yourself is an occupational hazard of being a scientist.

You are not the first or the last scientist to doubt what you are doing will be of any importance.

Believe me, I have had my share of nail-biting doubts that kept me awake for days on end and I failed many times in my experiments.

I can't explain it, but it is like science chose me rather than I chose science . I don't know why that is.

But please remember, you are not alone.