PROTEIN UPTAKE IN AMOEBAE 617 



Discussion 



Allen: In the case of tissue cells it is possible to see especially in time lapse 

 movies that the formation of pinocytotic vesicles is dependent upon the formation 

 of pseudopodia, what appears to be a short of "chewing" movement of the 

 hyaloplasmic ruffles of tissue cells. I wonder if there is the possibility that the 

 same might actually take place in amoeba and have escaped notice. Have you 

 looked into this ? 



Marshall: We have looked into this, but have not seen in amoebae quite the 

 process you describe, nor have others who have studied this more thoroughly, I 

 believe. I would only agree with the point made by Dr. Holter in his review: 

 different cell types show different morphological patterns of uptake. And even in 

 one cell type, the amoeba, different agents invoke different responses (as Chapman- 

 Andresen has shown). I don't know whether we can equate all forms of pinocytosis ; 

 differences exist, but these may be less important than the general similarities. 



Porter: What do you regard as the source of the hydrolytic enz\TTies acting in 

 these vesicles ? Is it possible that they are contained in some of the smaller vesicles 

 that you see associated with the surface of the microvesicles ? 



Marshall: It is possible. We don't really know which way the microvesicles 

 are going. All those we see clustered around a big vesicle, in micrographs, contain 

 something which in density and texture closely resembles the substance within 

 the larger vesicle. Also, the microvesicles are found sometimes in a row, like a 

 string of pearls attached at the end to the larger vesicle. From these points, it 

 seems more likely that they are being detached from the larger vesicle, but I agree 

 that this sort of evidence by no means settles the question. We must still say that 

 transport in and out of this chamber may be "transmembrane" transport in the 

 strict sense, or may be achieved by the addition or subtraction of microvesicles. 



GoLD.'XCRE : I am interested to see that there is no evidence of very tight packing 

 of your ferritin molecules on the outside of the membrane which might indicate 

 a tendency to expand the outside and thus cause a mechanical invagination of the 

 vesicles. I wonder if you can see in any of your pictures evidence of that or anything 

 else which might suggest a mechanism? 



M.^rshall: There is evidence suggesting expansion of the coat substance. 

 Schumaker's kinetic studies, you may recall, showed a brief second stage of 

 protein uptake, as though the first binding led to the appearance of new binding 

 sites. The ferritin work points in the same direction ; when an amoeba is treated 

 in the cold and washed, the entire surface is covered by ferritin initially. As the 

 cell warms up it changes shape, the coloured material accumulates in smaller 

 regions, and the greater part of the cell surface becomes clear. A few minutes after 

 clearing, the new surface will again bind ferritin or methylferritin. This implies 

 that new surface material, and probably new membrane as well, is formed very 

 rapidly. The amoeba surface is a dynamic, continually renewed structure, and we 

 think this is particularly so at the tips of advancing pseudopodia. All this can be 

 seen with living cells in the light microscope. At the lev'el of fine structure, it may 

 be explained by a fusion of microvesicles into the original membrane, the contents 

 of the microvesicles becoming new coat substance. We have seen occasionally 

 something to suggest this in electron micrographs, and hope to find out if it is true. 



