The Dual Characteristics of Light-Induced Cryptochrome 2, Homo-oligomerization and Heterodimerization, for Optogenetic Manipulation in Mammalian Cells

Abstract

The photoreceptor cryptochrome 2 (CRY2) has become a powerful optogenetic tool that allows light-inducible manipulation of various signaling pathways and cellular processes in mammalian cells with high spatiotemporal precision and ease of application. However, it has also been shown that the behavior of CRY2 under blue light is complex, as the photoexcited CRY2 can both undergo homo-oligomerization and heterodimerization by binding to its dimerization partner CIB1. To better understand the light-induced CRY2 activities in mammalian cells, this article systematically characterizes CRY2 homo-oligomerization in different cellular compartments, as well as how CRY2 homo-oligomerization and heterodimerization activities affect each other. Quantitative analysis reveals that membrane-bound CRY2 has drastically enhanced oligomerization activity compared to that of its cytoplasmic form. While CRY2 homo-oligomerization and CRY2-CIB1 heterodimerization could happen concomitantly, the presence of certain CIB1 fusion proteins can suppress CRY2 homo-oligomerization. However, the homo-oligomerization of cytoplasmic CRY2 can be significantly intensified by its recruitment to the membrane via interaction with the membrane-bound CIB1. These results contribute to the understanding of the light-inducible CRY2-CRY2 and CRY2-CIB1 interaction systems and can be used as a guide to establish new strategies utilizing the dual optogenetic characteristics of CRY2 to probe cellular processes.

Keywords: CRY2-CIB1 dimerization; cryptochrome 2; light control; oligomerization; optogenetics.

Figure 1

Membrane-bound CRY2 exhibit drastically enhanced oligomerization upon blue light stimulation. The cells were illuminated with intermittent 200 ms blue light pulse at every 5 s. (a) Cytoplasmic CRY2-mCh forms a few clusters upon blue light activation (yellow arrows). (b–d) Blue light stimulation induces dramatic cluster formation when CRY2 is tethered to various cellular membranes such as the ER membrane (b), plasma membrane (c), and mitochondrial outer membrane (d). (e) A light-insensitive mutant CRY2(D387A) localized on the ER membrane (CRY2(D387A)-mCh-Sec61) does not form clusters under blue light activation. (f) Spatial control of CRY2 clustering in the specific subcellular region (marked with a blue circle). Scale bars, 10 µm.

Figure 2

Light-induced CRY2 clusters are dynamic and reversible. COS-7 cells were transfected with CRY2-mCh-Sec61. (a) CRY2 clusters grow significantly in number, size, and intensity over several minutes after a single 2 s pulse blue light activation. Some clusters merge together to form higher order oligomers (yellow arrows). (b) CRY2 clusters, clearly visible at t = 5 min and less at t = 10 min, completely dissociate back to the diffusive ER distribution approximately 20 min after the single 2 s pulse blue light activation. Scale bars, 10 µm.

Figure 3

CRY2 oligomerization depends on the expression level of CRY2 and occurs readily at low level of blue light stimulation. Cells were activated with a single 2 s blue light pulse unless noted otherwise. The images were taken at 150 s after the blue light pulse. (a) A transfected cell with low CRY2-mCh-Sec61 expression has many CRY2 clusters, but the diffusive background is still visible after 150 s. (b) A transfected cell with high CRY2-Ch-Sec61 expression shows dramatic cluster formation. The diffusive background is invisible after 150 s. (c) Quantification of CRY2 cluster formation using an automatic Matlab algorithm. The peak of the cluster mass clearly shows that high CRY2 expression induces more CRY2 oligomerization compared to that of cells with low CRY2 expression. (d) The CRY2 oligomerization activity does not show significant reduction when the total blue light power is reduced by 300 times. (e) The CRY2 cluster formation is visibly decreased (clusters shown as yellow arrowheads) when the blue light intensity is reduced by 1200 times compared with normal stimulation condition. (f) Quantification of CRY2 cluster formation under different blue light intensities and exposure duration conditions. Error bars represent the standard deviation of the mean. Scale bar, 10 µm.

Figure 4

CRY2 oligomerization and CRY2-CIB1 heterodimerization coexist in the same system. Both CRY2 and CIB1 were tethered to the plasma membrane via a Caax motif. Before blue light stimulation, both CRY2 (red channel) and CIB1 (green channel) are homogeneous on the plasma membrane. Upon blue light illumination (200 ms blue light pulse every 5 s), CRY2 forms numerous bright clusters on the membrane. CIB1 is also found to accumulate in the same clusters. Scale bars, 10 µm.

Figure 5

CRY2 oligomerization on the ER membrane can be differentially suppressed by CRY2-CIB1 heterodimerization. Cells were illuminated with a single blue light pulse of 2 s. (a) Cotransfection of COS-7 cells with GFP-CIB1 and CRY2-mCh-Sec61 does not visibly affect CRY2 cluster formation on the ER membrane. (b) Coexpression of RAF-GFP-CIB1 and CRY2-mCh-Sec61 moderately reduces CRY2 cluster formation. The number and the intensity of clusters are reduced as compared with cells singly transfected with CRY2-mCh-Sec61. (c) Coexpression of GFP-BICDN-CIB1 and CRY2-mCh-Sec61 completely blocks CRY2 cluster formation, where no CRY2 cluster is visible after exposure to blue light stimulation. (d) Quantitative analysis of cluster formation in COS-7 cells cotransfected with CRY2-mCh-Sec61 and various CIB1 plasmids shows that CRY2 oligomerization can be suppressed at variable degrees. Error bars represent standard deviation of the mean. Scale bars, 10 µm.

Figure 6

Cytosolic CRY2 oligomerization can be drastically enhanced through CRY2-CIB1 heterodimerization and subsequent recruitment to the cell membrane. Cells were illuminated with 200 ms blue light pulse every 5 s. (a) A COS-7 cell cotransfected with CRY2-mCh and CIB1-GFP-Caax shows diffusive cytoplasmic CRY2-mCh and homogeneous plasma membrane-localized CIB1-GFP-Caax before blue light stimulation. After blue light exposure, CRY2-mCh is recruited to the plasma membrane and forms large clusters as shown in the red channel. In the green channel, CIB1-GFP-Caax shows the same cluster pattern as CRY2. (b) A COS-7 cell cotransfected with CRY2-GFP and CIBN-mCh-Miro1. Before blue light, CRY2-GFP is expressed in the cytoplasmic form. The partial colocalization seen between CRY2-GFP and mitochondria in the first imaging frame is due to the fact that blue light was used to collect the fluorescence signal in the green channel. After blue light, CRY2 clusters on the mitochondria well colocalize with the appearance of CIB1-mCh-Miro1 clusters in the same locations (yellow arrows). Scale bars, 10 µm. Insets show enlarged images as indicated by the dotted lines. Inset scale bar, 5 µm.

Figure 7

Proposed mechanism of CRY2-CRY2 oligomerization and CRY2-CIB1 heterodimerization. (a) Blue light induces conformational changes in CRY2, with CRY2-CRY2 binding and CRY2-CIB1 binding occurring at different CRY2 sites. Cytoplasmic CRY2 has low oligomerization activity due to random (unaligned) protein orientation. (b) CRY2 bound on the lipid membrane has enhanced oligomeric activity likely due to preferred parallel orientation for CRY2-CRY2 binding. (c) CIB1 linked with a bulky protein domain such as BICDN can suppress CRY2 oligomerization due to steric blocking of the CRY2-CRY2 binding site. (d) Cytoplasmic CRY2 can be recruited to the cell membrane by binding to membrane-linked CIB1, which significantly enhances CRY2 oligomerization.