Mimicking Morphogenesis in the Lab

Morphogenesis is the name given to the intricate process through which complex organs and tissues are created during the development of the fetus in-utero.  With the stunning and rapid advances made in the areas of biochemistry, molecular biology and genetics especially in regard to stem cells – stem cells are the pluripotent cells that have the inherent capacity to differentiate into a wide variety of specialized tissue cells -, a significant amount of information has been accumulated regarding the molecular mechanisms behind this process. 

   

In fact, there have been many recent examples of stem cells that have been induced to initiate morphogenesis in a laboratory setting (in vitro).  These studies have been outlined in a review article by Drs. Toshiro Sato and Hans Clevers from the Department of Gastroenterology at the Keio University School of Medicine in Tokyo, Japan that appeared in the journal Science, Vol. 340. June, 2013.  The example the reviewers cite came directly from their own efforts.  In their studies, they have successfully initiated morphogenesis in the laboratory in three-dimensional human cell cultures that resulted in the elaboration of what they refer to as, “Mini-Guts.”

One of the reasons they chose the small intestine as their in vitro model is because this organ has a higher self-renewal rate than any other mammalian tissue.  In fact, all the cells within the small intestine are renewed within five days.  The reason for this remarkable rate is due to the activity of a particular stem cell.  This stem cell type possesses a unique protein receptor on its surface, Lgr5, that binds preferentially with R-spondins a type of protein essential to the process of morphogenesis.  These Lgr5+ cells differentiate into a host of cell types that constitute a healthy and functional small intestine.  In addition, these Lgr5+ cells persist throughout the entire life of the organism.

To accomplish the successful in vitro production of mini-guts- epithelial organoids - that retain the identity and properties of the original tissue, the investigators used  Matrigel that is, in fact, a laminin and collagen-rich matrix that simulates the structural components of the basal lamina of the small intestine.  Additional factors and components that represent the minimal requirements for stem cell growth were also added to this matrix.

This impressive accomplishment has profound implications for the study of disease processes.  For example, it is now feasible to use this same methodology to produce mini-guts from cells derived from patients with adenomas and colorectal cancers.  These organoids could then be studied alongside of organoids derived from normal tissue.   Such studies could prove invaluable in understanding and delineating the etiology of these illnesses. 

The Fate of Incorrectly Folded Proteins

Cellular proteins play many diverse and essential roles in the living cell.  The roles of these proteins range from providing structural integrity and function in muscle, as an example, to assisting in the many chemical reactions that are essential to ordinary metabolism.  They also play a fundamental role in maintaining homeostasis throughout the entire organism.  Each cell contains a rich population of diverse proteins numbering literally in the thousands.  Given this reality, it is essential that these proteins function normally.

Ribosomes are the organelles – the term literally means little organs -that function as the site for protein manufacture within the cell.  The blueprint for the structure of each unique protein is specified by the cell’s DNA.  Once the newly formed protein is complete, it is released from the ribosome and enters the cellular cytoplasm.  Upon release from the ribosomal surface, the nascent protein must fold into a precise configuration in order to attain full functionality.  This folding process is spontaneous.  However, the many diverse macromolecules that fill the cytoplasmic intracellular environment can impose a serious impediment to this folding process.

 

To meet this challenge, a sophisticated “chaperone” system is designed to reduce unfavorable interactions with the cytoplasmic environment in order to enhance the likelihood of successful folding.  It does this through a repetitious series of binding and release.  If these repeated attempts fail, evidence from scientific investigations has shown that the folding process is ultimately terminated for it may pose a threat to cellular energy resources and increase the abundance of toxic reactive oxygen species.

The nature of this termination mechanism – using growing yeast cells as the model organism – has been studied in detail by Dr. Chenchao Xu and his colleagues from the Temasek Life Sciences Laboratory at the National University of Singapore.  From their studies, they have shown that the folding process is terminated by a specialized pathway that utilizes the modification of the unfolded protein by an enzyme-mediated chemical reaction known as  o-mannosylation involving  the transfer of mannose – a sugar – to the serine – an amino acid – residue of the protein.   This modification was shown to disable folding entirely.

 

Finally, the authors of this study propose that the function of this mechanism designed to thwart repeated folding attempts is to end apparently futile chaperone-directed folding.  As mentioned previously, repeated and unsuccessful cycles of folding can seriously impinge upon cellular energy reserves.  The fate of unfolded proteins is their ultimate degradation.

This kind of study helps to elucidate complex cellular mechanisms and highlights the importance of maintaining homeostasis within the cellular environment.