ASCB Celldance 2016 — “Discovery Inside Living Cells in Multicellular Organisms” Roberto Weigert
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ASCB Celldance 2016 — “Discovery Inside Living Cells in Multicellular Organisms” Roberto Weigert

A cell biologist would love to see what happens
inside the cell. The discovery of the green fluorescent protein
and improvements in light microscopy have brought new and exciting discoveries on a
yearly basis. Still, we always ask ourselves how do cells
behave in their native environment? They are surrounded by thousands of different
cells, by blood vessels that provide them nutrients, and by the extracellular matrix
that provide support. We now have the ability to look at them in
living tissues. In our lab, we have developed ways to image
subcellular processes inside the cells of several tissues. We call this approach: Intravital SubCellular
Microscopy or iSMIC. These videos show the dynamics on endocytosis
in the kidney and liver. It is like discovering a new world. Every day we go to the microscope, and we
have the opportunity to unravel new biology and ask new basic questions. Our main interest is to understand how cellular
membranes change shape during biological processes. This is called membrane remodeling. And we want to understand how certain proteins
provide mechanical forces to deform membranes. One of the processes that we study is protein
secretion in exocrine organs. When you eat, the pancreas and the salivary
glands secrete all the enzymes that you need to digest food. These enzymes are stored in large vesicles
that fuse with the plasma membrane. We found that after the enzymes are released
outside the cells, the membranes of the vesicles are inserted and absorbed into the plasma
membrane. We call this process integration. This process cannot be reproduced in cell
culture. In this movie, you can see secretory vesicles
in the salivary glands — highlighted by the red arrows — that fuse and are integrated
into the plasma membrane — the blue line. This is a beautiful example of membrane remodeling. So what drives the integration? We found that actin filaments are important. They form a cage around the vesicles that
contracts and pushes the membranes. The contractions are driven by an actin-based
motor called myosin II. Myosin II, which also forms filaments, binds
to F-actin and generates tension. Like the fingers squeezing this ball, we will
show you how we studied this process. In order to see the actin appearing on the
vesicles, we use mice engineered to express a fluorescent marker for filamentous actin
— in green — and a marker for the plasma membrane — in red. If you focus on the inset in this movie, you
can see the secretory vesicles in the pancreas that fuse with the plasma membrane and become
rapidly coated with F-actin before their integration. When we take away actin using a drug that
does not allow the formation of the filaments, the vesicles do not integrate into the plasma
membranes. Instead they begin to grow and get stuck at
the plasma membrane. We can also image how myosin II jumps on the
granules after the actin filaments are assembled. In this video, we used a mouse that expresses
fluorescent actin in red and fluorescent myosin II in green. The vesicles integrate into the plasma membrane
after myosin II is recruited. But in mice engineered to not express myosin II, the integration is stopped. Here’s this contractile machinery also used
in other physiological or pathological processes. We are beginning to investigate this question. For example, during cell motility. This is an immune cell that is moving inside
the blood vessel in breast tissue. The green florescence is again myosin II that
is recruited in different areas of the plasma membrane to drive this complex motion to contractions. Or during wound healing. These are neutrophils in a mouse that express
a marker for the actin filaments and that crawl inside a wound to help repair the injured
skin. The membranes of the cells are constantly
remodeled as the cells move. Or in tumors. These are a few examples of what can be accomplished
with Intravital SubCellular Microscopy. This approach is opening the door to investigate
cell biology under both physiological and pathological conditions in multicellular organs. This is a dream coming true.

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