Flat clathrin lattices are linked to metastatic potential in colorectal cancer

Background

Flat clathrin lattices (FCLs), clathrin sheets, clathrin-coated plaques – these are just some of the various names for a unique morphological form of clathrin, an essential protein recruited during clathrin-mediated endocytosis (CME) used for the internalization of cell surface receptors. FCLs are characterized by their planar, honeycomb-like lattices found on both the dorsal and ventral membranes of cells. FCLs were first depicted as a mere transition stage during CME, between the stages of early recruitment of clathrin to the plasma membrane and formation of a mature clathrin-coated vesicle. With the advent of high-resolution microscopy and reliable automated tracking software, it has become clear that FCLs play more diverse roles than we would have imagined. Despite their roles in CME, FCLs provide a platform for growth factor and adhesion receptor signaling, sense substrate rigidity to control cell signaling and proliferation, and support cancer cell migration. Growing interest in the dysregulation of endocytic proteins and aberrant clathrin organization in cancer has led to a recent study investigating the potential link between FCLs and cancer metastasis. 

Key findings

Researchers from an inter-laboratory collaboration between the Katholieke Universiteit Leuven, Instituto de Salud Carlos III, and Max Planck Institute for Polymer Research are the first to discover a direct link between the composition of FCLs and metastatic potential of colorectal cancer (CRC) cells. CRC was used as the metastatic model as the authors previously established that clathrin-mediated endocytic proteins are among the most dysregulated molecules in highly metastatic CRC compared to poorly metastatic CRC. 

The authors first confirmed that FCLs are prevalent at the ventral membrane of highly metastatic CRC cells. Two isogenic cell models of CRC metastasis were used, KM12 and SW, representing both poorly and highly metastatic versions of CRC metastasis. These cells expressed a fluorescently tagged version of one of clathrin’s proteins that make up its triskelion structure, termed clathrin light chain A. With confocal microscopy, the researchers found that clathrin largely appeared as diffraction-limited, dynamic clathrin-coated structures (CCSs) in poorly metastatic KM12 cells, whereas clathrin additionally appeared as large, elongated FCLs in highly metastatic KM12 cells (Figure 1). These FCLs were not only prevalent in highly metastatic CRC cells, but extremely stable, lasting from several minutes to more than 8 hours. 

 

While the general morphology of clathrin structures can be observed with confocal microscopy, different techniques are required to analyze the composition of clathrin across the ventral membrane of cells. To this end, the authors developed an algorithm that accurately classifies individual clathrin clusters as CCSs or FCLs, based on photoactivated localization microscopy/total internal reflection fluorescence microscopy (PALM/TIRF) single-molecule localization microscopy (SMLM) images of cell membranes. Together, these microscopy techniques eliminate background fluorescence from the cytoplasm and reduce the error in distinguishing CCSs that are spatially close together. The algorithm itself is distinct from current algorithms for classifying CCSs, as it globally thresholds parameters that characterize CCSs, eliminating the need to introduce excess user bias into the analysis by thresholding these parameters individually. 

Using the algorithm, the researchers observed that the areas of FCLs vary in different cell models, but on average, FCLs are approximately 6 times larger than CCSs. Notably, large, interconnected networks of FCLs associated with highly metastatic KM12 and SW cells, while small, discrete clusters of FCLs associated with poorly metastatic cells (Figure 2A). Comparing the ratio of FCLs to CCSs, the authors found that this ratio was significantly higher for both highly metastatic KM12 and SW cells compared to their poorly metastatic counterparts (Figure 2B). Thus, greater FCL composition is associated with increased metastatic potential in CRC. 

 

What factors contribute to creating such apparent differences in clathrin topology between poorly and highly metastatic CRC cells? Although the authors investigated the role of possible molecular contributors, such as those involved in CME (clathrin light chain A, clathrin heavy chain 1, AP2 subunit α2), they found no apparent contribution based on the expression of the proteins tested here. Due to other caveats that may contribute to the formation of FCLs, such as variability in protein expression between cell models or the involvement of mechanobiological cues, it remains an open question why FCLs are more prevalent in highly metastatic CRC cells.

Why this work is important

I chose this preprint because it highlights a largely unexplored area of mechanobiology in cancer. Much emphasis on the physical interaction between cancer cells and their microenvironment has been on focal adhesions. This preprint provides a promising starting point for understanding the role of FCLs in cancer.

From a therapeutic perspective, CRC is the third most commonly diagnosed cancer worldwide, with approximately 20% of its patients presenting with metastatic CRC at diagnosis and 25% who will later develop metastatic CRC. While CRC metastasis is typically limited to a few organs, such as the liver, lung, and lymph nodes, and is therefore amenable to complete surgical removal, micrometastases of CRC cells to other areas of the body are common. Finding the key characteristics that differentiate highly metastatic CRC cells from poorly metastatic CRC cells is needed for the development of targeted therapies, which can ultimately improve patient outcomes by preventing or treating metastatic CRC.

Questions and future directions for the authors

• It is possible that FCL formation on the ventral membrane varies as cells spread and encounter different stages of the cell cycle. When were the single-time point images of FCLs in cells taken? 
• Did you notice any signs of loss of fluorescence signal while imaging clathrin dynamics for extended periods (e.g. over 8h)
• Would it be beneficial to see whether knockdown of each of the three endocytic proteins tested for their expression levels (CLTA, CLTC, AP2A2) has an effect on FCL formation?
• It would be intriguing to compare FCL formation in metastatic CRC cells with normal colon cells, as well as in cells seeded in 3D surfaces such as collagen. What are your plans moving forward from your findings presented in this preprint? 

 

Posted on: 6 April 2023

doi: https://doi.org/10.1242/prelights.34290

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