Distinct nanoscale organizations of mucins and trans-sialidases in Trypanosoma cruzi

Reviewer #1 (Public review):

Summary:

Escalante et al. employ super-resolution microscopy to achieve a clearer, nanoscale view of how trans-sialidases and mucins are organized on the Trypanosoma cruzi parasite membrane. Comparing the experimental data using clustering analysis with model-based simulations, they report two kinds of organizational states describing the non-uniform distribution of these two proteins: a segregated state where mucins and trans-sialidases form spatially distinct nano-clusters, and a non-clustered state where they share a proposed fibrillar network with more ordered, shorter-than-random separation distances. They also look at the oligomerization states of the two proteins to try and propose a mechanistic basis for the observed distributions.

Strengths:

The in-depth analysis of the distributions of both proteins coupled with model-based simulations brings out new insights into organizational principles underlying protein distribution on the membrane surface. The ability to resolve shorter-than-random separation distances even in the non-clustered state is to be highlighted and is a key take-away from this manuscript.

Weaknesses:

The authors propose the oligomeric state of mucins compared to the non-oligomeric trans-sialidases as a basis for explaining the distinct organization of these proteins. Although this hints at how segregation may occur, it does not inform us of how the more ordered non-clustered state could co-exist with the clustered segregated state and warrants further investigation.

Overall, the analytical framework applied in this study to elucidate organizational principles for the non-uniform distribution of proteins can potentially be used in a wide-range of contexts across different organisms and systems. This study also lays the groundwork to understand mechanisms that spatially regulate how trans-sialidases act on their substrates. Going forward, it could be very interesting to look at how different kinds of mucins and trans-sialidases are organized with respect to one-another and amongst themselves. Also, the development of tools to observe the dynamics of these proteins live will likely provide further insights into the mechanism.

Reviewer #2 (Public review):

The manuscript describes a numerical analysis of the domains of the T. cruzi cell surface containing different proteins. It has the potential to be of great interest.

I do not have the expertise necessary to comment on the image collection or analysis.

I have one concern: the amount of manipulation of the cells prior to fixation; these were clearly stated in the methods, which is good.

My concern is whether these manipulations prior to fixation alter the observations. The 'Labelling sialic acid acceptors' involves >6 centrifugations and >90 minutes incubation in PBS prior to fixation, and the 'immunostaining' protocol involves cells 'extensively washed with PBS' prior to fixation. I would like to suggest that the authors do controls in which they compare the pattern of anti-SAPA staining under four conditions.

(1) Cells fixed in culture by the addition of paraformaldehyde to 4%, followed by blocking and PBS washes.

(2) Cells fixed in culture by the addition of paraformaldehyde to 4% and glutaraldehyde to 0.2% followed by blocking and PBS washes.

(3) Cells fixed by the 'labelling sialic acid acceptors' protocol.

(4) Cells fixed by the 'immunostaining protocol'.

Reviewer #3 (Public review):

Summary:

The authors present an innovative approach to tackle the lateral organization of mucins and trans-sialidases (TS) on the cell membrane of the organism Trypanosoma cruzi. By applying dual-color super-resolution microscopy (STORM), the authors report on a differential nanoscale distribution between mucins and TS on the cell membrane. Moreover, they find that 60% of mucins and TS are organized in nanoclusters with an inter-nanocluster distance following a random distribution. The remaining 40% of both proteins are organized in a non-random manner, and, using simulations, the authors claim that they are organized in rectilinear fibers.

Strengths:

The authors use dual-color STORM microscopy to unravel the protein nanoscale organization of mucins and TS on the cell membrane of Trypanosoma cruzi for the first time. They perform a dedicated analysis of the localizations and clustering of both proteins. Moreover, they perform, for every type of analysis on real data, simulations to compare their results for random organization. They also use an analysis approach together with simulations to propose that the lateral organization of both non-clustered proteins are within rectilinear fibers. They also complement their microscopy findings with BN-PAGE. Overall, the use of advanced microscopy techniques, corresponding data analysis and simulations is very solid and remarkable.

Weaknesses:

As the authors point out, they do not provide a molecular/biophysical mechanism explaining the non-random lateral organization of mucins and TS (both clustered and individual proteins).