John M. Robinson, Ph.D.

Professor Director, Office of Postdoctoral Programs 212/215 Hamilton Hall 1645 Neil Avenue Columbus  OH  43210-1218 robinson.21@osu.edu View CV

Education:
B.S., Alabama College, Montevallo, Alabama
Ph.D., Biology, Vanderbilt University, Nashville, Tennessee
Research Fellow in Pathology, Harvard Medical School, Boston, MA

Research Area:
Cell biology of phagocytic leukocytes, endothelium, and the placenta.

Placental Biology and Plasma Membrane Function

The placenta is a vital, if ephemeral, organ that facilitates the exchange of solutes and gases between the mother and developing fetus. The placenta also provides a barrier to the maternal immune system and produces hormones and growth factors that support pregnancy. In short, proper placental function is key for normal fetal development.

The inner aspect of the human placenta is covered by a unique cellular layer known as the syncytiotrophoblast (STB). The STB forms the interface between maternal blood and the placenta and fetus. Specifically, it is the apical plasma membrane of the STB that interacts with maternal blood. Even though this membrane is crucial to placental function, surprisingly little is known about it at the molecular level.

In order to study this membrane in greater detail, a highly enriched preparation of the apical plasma membrane of the STB was generated. Development of methods for obtaining highly enriched membrane from the STB was carried out in collaboration with Dale D. Vandré, Ph.D. from this department and William E. Ackerman, M.D, from the Department of Obstetrics and Gynecology here at OSU. Our method for generating highly enriched membrane preparation allowed us to carry out a proteomics profiling of this membrane by mass spectrometry. The mass spectrometry facility is housed in the Campus Chemical Instrumentation Center: http://www.ccic.ohio-state/MS/index.htm. For more information on placental proteomics, please refer to Professor Vandré’s webpage.

We identified a large number of proteins in this profiling study; many of these were heretofore unknown in the placenta. Notably, we discovered two members of the ferlin protein family, dysferlin and myoferlin. These proteins were previously known primarily from studies of skeletal muscle. We have shown, by immunofluorescence microscopy, that dysferlin is located primarily in the apical plasma membrane of the STB in placenta (Fig. 1). Cytotrophoblasts (CTB), the cells that give rise to the STB, lack detectable dysferlin in vivo. CTBs can be isolated from the placenta and when placed into cell culture lack dysferlin. However, these cells are capable of spontaneous cell-cell fusion and when fused they express dysferlin. Thus the in vitro system recapitulates the in vivo situation.

The significance of this finding is that dysferlin has been implicated in the repair of damaged plasma membranes in skeletal muscle. Experiments are underway to determine the mechanism of membrane repair in placental trophoblasts. This is likely to be an important in placental biology since the apical plasma membrane of the STB is significant membrane stress.

Recent publications describe our initial work in this area:

• Vandré DD, Ackerman WE 4th, Kniss DA, Tewari AK, Mori M, Takizawa T, Robinson JM. Dysferlin is expressed in human placenta but does not associate with caveolin. Biol Reprod 2007 Sep; 77(3):533-42.

• Robinson JM, Ackerman WE 4th, Kniss DA, Takizawa T, Vandré DD. Proteomics of the human placenta: promises and realities. Placenta 2008 Feb;29; (2):135-43.

Figure 1. Comparison of DYSF labeling in the apical and basal plasma membranes of the STB. Confocal microscopy images of immunolabeled cryostat sections of placenta were used to determine the fluorescence intensity associated with the apical and basal plasma membranes of the STB. In this representative image, the apical plasma membrane (double arrows) and the basal membranes (single arrows) are indicated. Low-level labeling of endothelial cells was present in this section; the asterisks (*) indicates the vessel lumen. Bar = 10 µm. From: Biol Reprod 2007 Sep; 77(3):533-42.

Figure 2. Syncytial formation by cytotrophoblasts in vitro. A) Isolated CTB that were cultured for four days and then prepared for the IFM localization of PLAP. Mononuclear CTB have fused together to form syncytial-like structures that express PLAP (green color). The fluorescence image DAPI-stained nuclei are indicated by blue color. Adjacent cells not yet fused with the syncytial-like structure do not express PLAP (arrows). B) The same cells shown in panel A with the DIC image merged with the fluorescence image of DAPI-stained nuclei to show the morphology of the syncytialized structure. Arrows serve as reference points.

C) Isolated CTB were cultured for four days and then prepared for the IFM localization of DYSF. Mononuclear CTB have fused together to form syncytial-like structures that express DYSF (green color). The fluorescence image DAPI-stained nuclei are indicated by blue color. Adjacent cells not yet fused with the syncytial-like structure do not express DYSF (arrows). 
D) The same cells shown in panel C with the DIC image merged with the fluorescence image of DAPI-stained nuclei to show the morphology of the syncytialized structure. Arrows serve as reference points.
E) Immunoblot analysis confirms the expression of dysferlin in cultured CTB. Lysates of cultured CTB (lane 1) and lysates of rat skeletal muscle (lane 2) were probed with the monoclonal anti-DYSF. Bands of the same apparent electrophoretic mobility were detected in cultured CTB and muscle.
F) Immunoblot analysis failed to detect the expression of CAV3 in cultured CTB. Lysates of cultured CTB (lane 1) and lysates of rat skeletal muscle (lane 2) were probed with a monoclonal antibody to CAV3. While present in skeletal muscle, CAV3 could not be detected in cultured CTB Bars = 20 µm. From: Biol Reprod 2007 Sep; 77(3):533-42.