Clinton T. Morgan

COMMD1 Forms Oligomeric Complexes Targeted to the Endocytic Membranes via Specific Interactions with Phosphatidylinositol 4,5-Bisphosphate

Copper metabolism Murr1 domain 1 (COMMD1) is a 21-kDa protein involved in copper export from the liver, NF-κB signaling, HIV infection, and sodium transport. The precise function of COMMD and the mechanism through which COMMD1 performs its multiple roles are not understood. Recombinant COMMD1 is a soluble protein, yet in cells COMMD1 is largely seen as targeted to cellular membranes. Using co-localization with organelle markers and cell fractionation, we determined that COMMD1 is located in the vesicles of the endocytic pathway, whereas little COMMD1 is detected in either the trans-Golgi network or lysosomes. The mechanism of COMMD1 recruitment to cell membranes was investigated using lipidspotted arrays and liposomes. COMMD1 specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the absence of other proteins and does not bind structural lipids; the phosphorylation of PtdIns at position 4 is essential for COMMD1 binding. Proteolytic sensitivity and molecular modeling experiments identified two distinct domains in the structure of COMMD1. The C-terminal domain appears sufficient for lipid binding, because both the full-length and C-terminal domain proteins bind to PtdIns(4,5)P2. In native conditions, endogenous COMMD1 forms large oligomeric complexes both in the cytosol and at the membrane; interaction with PtdIns(4,5)P2 increases the stability of oligomers. Altogether, our results suggest that COMMD1 is a scaffold protein in a distinct sub-compartment of endocytic pathway and offer first clues to its role as a regulator of structurally unrelated membrane transporters.

Chemical reprogramming of Caenorhabditis elegans germ cell fate

Small-molecules can control cell fate in vivo and may allow directed induction of desired cell types, providing an attractive alternative to transplant-based approaches in regenerative medicine. We have chemically induced functional oocytes in C. elegans adults that otherwise produced only sperm. These findings suggest that chemical approaches to therapeutic cell reprogramming may be feasible and provide a powerful platform for analyzing molecular mechanisms of in vivo cell reprogramming.

Phosphorylation state of a Tob/BTG protein, FOG-3, regulates initiation and maintenance of the Caenorhabditis elegans sperm fate program.

FOG-3, the single Caenorhabditis elegans Tob/BTG protein, directs germ cells to adopt the sperm fate at the expense of oogenesis. Importantly, FOG-3 activity must be maintained for the continued production of sperm that is typical of the male sex. Vertebrate Tob proteins have antiproliferative activity and ERK phosphorylation of Tob proteins has been proposed to abrogate “antiproliferative” activity. Here we investigate FOG-3 phosphorylation and its effect on sperm fate specification. We found both phosphorylated and unphosphorylated forms of FOG-3 in nematodes. We then interrogated the role of FOG-3 phosphorylation in sperm fate specification. Specifically, we assayed FOG-3 transgenes for rescue of a fog-3 null mutant. Wild-type FOG-3 rescued both initiation and maintenance of sperm fate specification. A FOG-3 mutant with its four consensus ERK phosphorylation sites substituted to alanines, called FOG-3(4A), rescued partially: sperm were made transiently but not continuously in both sexes. A different FOG-3 mutant with its sites substituted to glutamates, called FOG-3(4E), had no rescuing activity on its own, but together with FOG-3(4A) rescue was complete. Thus, when FOG-3(4A) and FOG-3(4E) were both introduced into the same animals, sperm fate specification was not only initiated but also maintained, resulting in continuous spermatogenesis in males. Our findings suggest that unphosphorylated FOG-3 initiates the sperm fate program and that phosphorylated FOG-3 maintains that program for continued sperm production typical of males. We discuss implications of our results for Tob/BTG proteins in vertebrates.

Mitosis-meiosis and sperm-oocyte fate decisions are separable regulatory events.

Germ cell fate decisions are poorly understood, despite their central role in reproduction. One fundamental question has been whether germ cells are regulated to enter the meiotic cell cycle (i.e., mitosis–meiosis decision) and to be sperm or oocyte (i.e., sperm–oocyte decision) through one or two cell fate choices. If a single decision is used, a male-specific or female-specific meiotic entry would lead necessarily toward spermatogenesis or oogenesis, respectively. If two distinct decisions are used, meiotic entry should be separable from specification as sperm or oocyte. Here, we investigate the relationship of these two decisions with tools uniquely available in the nematode Caenorhabditis elegans. Specifically, we used a temperature-sensitive Notch allele to drive germ-line stem cells into the meiotic cell cycle, followed by chemical inhibition of the Ras/ERK pathway to reprogram the sperm–oocyte decision. We found that germ cells already in meiotic prophase can nonetheless be sexually transformed from a spermatogenic to an oogenic fate. This finding cleanly uncouples the mitosis–meiosis decision from the sperm–oocyte decision. In addition, we show that chemical reprogramming occurs in a germ-line region where germ cells normally transition from the mitotic to the meiotic cell cycle and that it dramatically changes the abundance of key sperm–oocyte fate regulators in meiotic germ cells. We conclude that the C. elegans mitosis–meiosis and sperm–oocyte decisions are separable regulatory events and suggest that this fundamental conclusion will hold true for germ cells throughout the animal kingdom.

Laparoscopic Surgery for Para-esophageal Hernias

Surgical management of paraesophageal hernias has changed dramatically during the past two decades. Elective laparoscopic paraesophageal hernia repair is now the standard approach to the patient with a symptomatic paraesophageal hernia. The operation is technically challenging and requires numerous intra-operative and peri-operative considerations. Here, we will highlight the key points in the peri-operative and intra-operative management of paraesophageal hernias, emphasizing the operative goals of reduction of the hernia contents, excision of the hernia sac, a tension free repair with complete esophageal mobilization, crural repair, and fundoplication.

Systemic Chemotherapy for Resectable Hepatic Colorectal Metastases: Adjuvant, Neoadjuvant, or Not at All?

The contemporary management of hepatic colorectal adenocarcinoma metastasis relies on the multidisciplinary use of both local operative resection and systemic chemotherapy. This article will discuss the principles and data that are of relevance in determining the optimal way to combine these treatment modalities. In so doing, we will explore the issue of when and whether to administer chemotherapy for patients with resectable hepatic metastases.