Image Cross Correlation Spectroscopy Used To Study the Membrane Localization of BMPRIa and FGF1

Abstract

Bone morphogenetic protein 2 (BMP2) and fibroblast growth factor 1 (FGF1) are known regulators of osteoblast proliferation and bone growth. BPM2 is known to induce differentiation of osteoblasts from mesenchymal stem cell precursors. Signaling which controls this process is initiated by the binding of BMP2 to BMP receptor type Ia (BMPRIa), initiating the dissociation of the BMPRIa associated protein casein kinase II (CK2) at one of three binding sites. Selective blocking of CK2 association with specially designed CK2 blocking peptides (CK2.1, CK2.2, CK2.3) has led to both osteoblast and adipocyte differentiation, the balance of which is altered in the disease osteoporosis. BMP receptor signaling has been found to be dependent upon receptor relocalization and aggregation on the plasma membrane, with the membrane domain caveolae playing a particularly important, though poorly understood role. These dynamics differ in cells isolated from mice of varying bone mineral density. FGF1 is another regulator of bone growth. Studies have shown that FGF1 readily induces new bone formation and can prevent bone loss in an induced osteoporotic rat model. FGF1 is released under stressed conditions via a membrane associated complex and transported across the plasma membrane by the poorly understood nonclassical transport mechanism, which bypasses packaging and modification in the endoplasmic reticulum and Golgi apparatus. The membrane dynamics of both bone regulatory proteins were studied with the technique image cross correlation spectroscopy (ICCS). This imaging technique quantifies the spatial similarity of fluorescently labeled proteins on the plasma membrane, termed colocalization values. ICCS analysis of FGF1 with membrane associated release complex components phospholipase D (PLD) and annexin II at elevated temperature (42°C) found increased membrane association with annexin II but decreased association with PLD. FGF1 transport is clearly affected by temperature but further experiments are required to draw conclusions. ICCS analysis of osteoporotic mice stimulated with BMP2 and CK2 blocking peptides showed reduced shuttling of BMPRIa and ultimately less signaling. This reduced signaling could provide the explanation for bone loss with osteoporosis. Stimulation of myoblast C2C12 cells with BMP2 and CK2 blocking peptides for 24 hours showed no significant BMPRIa aggregation or membrane relocalization, indicating no signaling. However, there was significant membrane movement of CK2 in response to CK2.3 treatment. Because signaling has been shown in previous studies with 18 hours stimulation, it is uncertain what is occurring with 24 hour stimulation. The pathway may reset at this time, in preparation for future signaling. Further studies involving other membrane domains, as well as stimulation at more varied times are necessary.