Background

Cell migration is one of the many fundamental processes that occur during normal development and physiology (Theveneau and Mayor, 2013; Krause and Gautreau, 2014). Central to this process is the main plasma membrane branched actin generating module, comprising of four main components: the small GTPase protein Rac1 (Machacek et al., 2009), the Scar/WAVE complex (Davidson and Insall, 2011; Krause and Gautreau, 2014), the Arp2/3 complex (Goley and Welch, 2006), and actin. When cells receive a stimulus, such as a growth factor or chemo-attractant, these molecules and complexes work together to nucleate the branched actin network and push the plasma membrane forward, promoting cell migration. Interestingly, macropinocytosis (Swanson and Watts, 1995; Bloomfield and Kay, 2016), an evolutionarily conserved endocytic process, shares the same molecular machinery as cell migration. However, instead of pushing in the plane of the front-rear axis of the cell, local actin polymerisation pushes plasma membrane sheets forward or upward, to form cup-like structures that resolve into macropinocytic vesicles taken into the cell. Cells use macropinocytosis not only to uptake nutrients but also to traffic and organize different membrane receptors, such as integrins, to modulate their adhesion and invasion in cancer (Le et al., 2021).

In our recent paper published in the Journal of Cell Biology (Le et al., 2021), we utilised an image-based internalisation assay, to show that cells lacking CYFIP-related Rac1 Interactor (CYRI) proteins displayed a reduced macropinocytic uptake of dextran 70 kDa.

In this Bio-Protocol article, we describe a step-by-step protocol to detect and quantify the amount of dextran uptake in cultured adherent cells. The wet lab procedure is inspired by Commisso (Commisso et al., 2014), with modifications to simplify the method. We tried different types of dextran, including dextran tetramethylrhodamine (TMR) (Invitrogen, #D1818), dextran Texas Red (Invitrogen, #D1830), and dextran Fluorescein (Invitrogen, #D1822). We found only the last type resulted in clean and analysable data without the need for excessive washing, while both the dextran TMR and dextran Texas Red resulted in a high background with visible clumps of protein, even after multiple washes. We also omit the overnight starving steps, as we found that they were not essential and made no difference to the outcome, using our conditions and cells. This significantly reduced the length of time required for the assay. Since the method described here has been used quite commonly and successfully in the literature before, we focus more on simplifying the experimentation steps and improving the automation capability in the quantification step. We provide the full macro script that can be directly copied and pasted into your Macro window in ImageJ/Fiji (Schindelin et al., 2012). We also provide comments to explain in simple terms what each command in the script does, which could be very useful for those who have no background in coding, or who have just started their journey in programming. We believe this is something that is still missing in the current literature, where there are a lot of resources for image analysis, but many are not necessarily accessible or presented in understandable terms for everyone, particularly beginners. The image analysis pipeline presented here is also suitable for analysing any other intracellular signal, including but not limited to integrin internalisation (Le et al., 2021), transferrin, and other endocytic processes.