Proteins Affected by FP Tagging

In this project I analyzed the organization of both cytosolic and membrane attached proteins.

Among the cytosolic proteins I investigated, β-actin, amphiphysin, Munc18-1, and PIPKIγ showed significant difference when FP tagged (Figure 4-1). Only three membrane attached proteins were affected in different extent when fused to FPs.

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Cytosolic Proteins

PIPKIγ (phosphatidylinositol 4-phosphate 5-kinase type-1 gamma) concentrates at synapses where it acts as the major generator of PtdIns(4,5)P2 (Ishihara et al., 1998; Wenk et al., 2001) and controls major processes, such as actin remodeling, cell adhesion, and endocytotic vesicle transport (Di Paolo et al., 2002; Doughman et al., 2003; Krauss et al., 2006; Li et al., 2013). Pietro De Camilli and colleagues proved the 28-aa C-terminal tail in PIPKIγ is essential for its association with focal adhesions (Di Paolo et al., 2002). PIPKIγ showed a highly significant change in all three parameters investigated.

The detected spots of FP tagged PIPKIγ are ~60% bigger and both the peak intensity and the total intensity of the spots are ~3-fold higher. The isoforms α and β of PIPK have been recently shown to dimerize with important consequences on their enzymatic activity and interaction with other proteins and membranes (Hu et al., 2015; Lacalle et al., 2015). For PIPKIγ, such studies are missing but the results herein suggest that FP tagging induces the formation of higher order oligomers. Even though PIPKIγ has been used so far only as N-terminal GFP fusions (Di Paolo et al., 2002), the C-N-terminal chimera used in this study has shown proper localization. Interestingly, the PIPKIγ interaction partner AP-2µ, which binds and clusters around the endocytotic cargo (Krauss et al., 2006; van den Bout and Divecha, Figure 4-1 Overview of significance in t-tests for the investigated proteins

Graph depicting the results in Student’s t-test for the three parameters that were assessed. If the difference between the FP-tagged and the non-tagged version of the protein of interest is statistically significant a rectangle filled in with blue-green (for spot size), in red (for peak intensity), or in yellow (for the total intensity) is shown above the protein name.

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113 2009), did not show any significant differences in the protein assemblies it forms as an FP chimera.

Cytosolic proteins β-actin and Munc18-1 have spots brighter by ~50-60% upon FP tagging, both for peak intensity and total peak intensity. Yet, their spot size does not differ significantly. This finding suggests that FP fusions associate in higher numbers into multimers that retain more-or-less similar sizes like their non-tagged variants.

β-actin has been widely used for in vivo and in vitro experiments as an C-terminal chimera with FP (Hodgson et al., 2000; Guo et al., 2007; Hamamoto et al., 2010). This protein is a major component of the cytoskeleton, forming microfilaments by binding to four other actin molecules (Holmes et al., 1990; Chen et al., 2000). So, for β-actin, it is easy to imagine that the FP tag can modify the geometry of β-actin assembly into oligomers, leading to the observed differences. The molecular arrangements formed by actin were shown to be very sensitive to sample preparation. For example, the periodical cytoskeleton formed by actin along the axon could be observed either using in vivo labeling techniques in STED (D’Este et al., 2015) or very careful embedding procedures in STORM (Xu et al., 2013).

Munc18-1, also known as syntaxin-binding protein 1, is a neuronal protein involved in synaptic transmission, especially vesicle docking and priming (Verhage et al., 2000; Deák et al., 2009). It has also been shown to bind to syntaxin 1 with high affinity and mediate its transport to the cell membrane. In addition, Munc18-1 regulates the activity of syntaxin 1 (Toonen et al., 2005; Gerber et al., 2008) and also binds the exocytotic SNARE complex (Deák et al., 2009; Meijer et al., 2012). For the Munc18-2 isoform, a point mutation has been shown to induce dimerization without affecting the ability of this protein to bind its interaction partner syntaxin 11 (Hackmann et al., 2013). No evidence for Munc18-1 dimerization or multimerization exists. However, the ~50% increase in spot brightness detected here indicates that the FP tag may increase the self-association of Munc18-1.

Amphiphysin is a protein which associates with synaptic vesicles and presynaptic membranes (Lichte et al., 1992). It contains an N-terminal BAR (Bin-Amphiphysin-Rvs) domain through which it interacts with membranes and induces their tubulation (Takei et al., 1999). The BAR domain can induce the dimerization of amphiphysin (Peter et al., 2004).

The amphiphysin isoform 1 (brain-specific) and isoform 2 (widely distributed) are also known to form heterodimers (Wigge et al., 1997) and homodimers mediated by the BAR domain (Peter et al., 2004). Amphiphysin 1 (mentioned throughout this work as amphiphysin) formed spots dimmer by ~20% when FP tagged, both in terms of peak

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intensity and total intensity. As the spot size is not affected, one possible explanation would be the association of fewer amphiphysin molecules in the presence of an FP tag, while retaining the overall size of the assembly remain unchanged. One plausible cause for this observation is the steric hindrance imposed by the FP tag.

Membrane Proteins

Among the membrane-attached or integral proteins, VAMP4 exhibits the biggest differences: its FP chimera forms assemblies smaller by more than 20% and dimmer by

~50%. A similar construct to the one used in this project, VAMP4-GFP, was shown to cycle between the cell membrane, recycling endosomes, and trans-Golgi network due to an N-terminal motif (Zeng et al., 2003; Tran et al., 2007). It is currently not known if VAMP4 associates into clusters like other SNARE proteins have been shown to do (Sieber et al., 2007; Halemani et al., 2010; Bar-On et al., 2012). But these results indicate that the presence of an FP tag inhibits VAMP4 association. In constrast to constitutively expressed VAMP4, the neuronal VAMP2 did not show any significant differences in any of the investigated parameters.

Another SNARE protein, Vti1a-β, forms somewhat bigger assemblies (by ~20%) when FP-tagged, without modifications in spot brightness. This points to a possible expansion of the protein assembly to accommodate the FP tag, without modifying the number of protein molecules per cluster. Vti1a-β is a brain-specific splice variant enriched in small synaptic vesicles (Antonin et al., 2000). Vesicles containing Vti1a participate in spontaneous fusion (Ramirez et al., 2012). However, no data is available with regard to its clustering behavior.

Interestingly, among the four types of syntaxin included in this study, only one of them, syntaxin 6, showed a significant difference in the total intensities of the detected spots but not of the spot size or the peak intensity. This could be the result of very small modifications in the organization of the assemblies formed by syntaxin 6. Such small intensity differences can be detected only using the total intensity analysis, which is more sensitive to dimmer spots. It is worth to note that syntaxin 7 also showed big differences in the parameter values when FP tagged, but these were not significant due to the large standard error of the mean.

Interestingly, syntaxin 13 (also known as syntaxin 12), a binding partner of syntaxin 6, is not affected by FP tagging. Syntaxin 1, a neuronal SNARE protein found on the plasma membrane, did not show differences in the clusters it formed in the presence or absence of a C-terminal YFP tag neither in STED nor in GSDIM. Possible reasons why only syntaxin 6 is more susceptible to FP tagging than the other syntaxins are the sizes of the transmembrane

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115 domains and the vesicular/extracellular exposed tails. According to Uniprot (www.uniprot.org), syntaxins 6, 7 and 13 all have transmembrane domains with 21 residues, while syntaxin 1 has 23. If the residues of the cytoplasmic tail are taken into consideration, then syntaxin 6 has no exposed residues in its tail, whereas syntaxin 7 and syntaxin 13 have 2 and 3, respectively. Hence, tagging with FP may induce stronger perturbations in syntaxin 6.

In principle, larger protein assemblies should exhibit also a higher total intensity, and vice-versa. This is indeed the case for PIPKIγ and VAMP4, the two proteins that showed the most pronounced differences (Figure 3-20). For the rest of the proteins, only small but significant differences in either the size or the intensity were observed. A possible explanation for this is that fluorescent dye molecules can undergo self-quenching in tightly packed protein clusters (Saka et al., 2014a). This observation is in line with the case of Vti1a-β, which forms bigger protein assemblies in the presence of an FP tag, but no increase in total fluorescence intensity is detected. In contrast, loose protein assemblies would describe protein clusters that are dim but not very dense. So they can accommodate more proteins or lose them without changing their size. If all other technical and conceptual biases are removed, this would be the case for the rest of the proteins that show significant differences only in their spot intensity profile (β-actin, amphiphysin, Munc18-1, and syntaxin 6)

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