Cells missing a certain protein on their surface can’t move normally, UConn researchers report in Science Signaling.
The research could give insight into how cells move and repair wounds in normal tissue, as well as how cancer spreads through the body.
Cells are the body’s workers, and they often need to move around to do their jobs.
Frequently, a cell will move through a tissue – say, the wall of a blood vessel – the way a rock climber scales a cliff, using a protein called integrin to grab onto a spot and pull itself in that direction.
How is integrin activated?
Integrin activation is an important mechanism through which cells regulate integrin function by manipulating the ligand affinity of integrins spatially and temporally.
Structural and functional studies suggest that integrins can exist in different ligand affinity states – low, intermediate and high .
Crystal structures have revealed that integrin heterodimers, occur in an inactive, bent V-shape with the head close to the membrane-proximal regions of the legs , maintained by the α/β salt bridge at the inner membrane region and helix packing of the transmembrane (TM) region.
his low affinity structure undergoes rapid, reversible conformational changes to increase ligand affinity, termed “activation”.
The Structural hallmarks of integrin activation are:
a) complete extension of the extracellular domains and
b) separation of the cytoplasmic leg domains .
This process facilitates integrin-mediated signaling, thus mechano-sensing and -transmitting.
Integrin can be activated from two directions, from the inside by the regulated binding of proteins to the cytoplasmic tails, and from the outside by multivalent ligand binding.
In either case, talin binding to the integrin β tails is an essential and the final common step .
Though the two processes are conceptually separate, they are mutually cooperative i.e one can lead to the other.
Some structural studies done with force application to mimic ligand/intracellular protein suggested that the combined action of these two events favor the transition from the closed, low affinity to a open, high affinity conformation of integrin
Activation leads to bidirectional signaling crucial in a variety of anchorage-dependent events such as adhesion, cell spreading, migration, polarity and organization of the ECM leading to physiological changes.
Here we discuss only well-established events that occur at the close proximity of integrin molecules localized on the plasma membrane.
A. Low-affinity integrin has an inactive, bent, conformation. B1 and B2. Inside-out integrin activation by cytoplasmic proteins or Outside-in integrin activation via ECM ligands both lead to complete extension of the extracellular domains. C. The hallmark of open, high-affinity activated integrin is separation of the cytoplasmic leg domains.
Inside-out signaling versus Outside-in signaling
Different signaling pathways can initiate integrin activation via:
Inside-out signaling
Signals received by other receptors foster the binding of talin and kindlin to cytoplasmic end of the integrin β subunit, at sites of actin polymerization. Substantial information on signaling pathway leading activation is available for integrin αIIbβ3.
Talin binds to integrin β-tail via F3 phospho-tyrosine binding (PTB) domain, a unique interaction with the membrane proximal (MP) region of the integrin (NPxY motif).
This permits competition between conserved lysine on talin and an aspartic acid on integrin α essential for α/β salt bridge disruption and sufficient for integrin activation.
Addition interactions through the basic patches in the FERM subdomain F2 helps to orient the β-subunit to promote spatial separation of the cytoplasmic domains .
Kindlin is also an essential co-activator of integrin and binds to a membrane distal NxxY motif on β-integrin via its FERM F3 subdomain [23].
A preceding threonine patch on integrins β1 and β3 that gets phosphorylated and a tryptophan on kindlin F3 are also required for binding. However, kindlins are not known to activate integrins on their own but may render integrin-specific effects .
The mechanism of crosstalk between integrin, talin and kindlin are not well established . However, substantial data on the order of their binding is available. Latest Findings Talin is recruited directly to FAs from the cytosol suggesting that it does not bind to free diffusing integrins outside FAs and also requires vinculin and F-actin for its activation .
Hence it is believed that only F-actin anchored talin at FAs bind free diffusing integrin promoting its activation. Talin can directly connect to actin while kindlin links through adaptors such as migfilin, filamin, FAK,VASP and α-actinin.
Outside-in signaling
Ligand binding to external domain causes conformational changes that increase ligand affinity, modify protein-interaction sites in the cytoplasmic domains and thence the resulting signals.
Besides conformational changes that extend integrin dimers , multivalent ligand binding leads to clustering of integrins, which in turn activates Src family of kinases (SFKs) by autophosphorylation.
SFKs phosphorylate tyrosines of the integrin cytoplasmic domain (NPxY motifs) and other proteins leading to
- control of ligand binding strength
- alteration of binding with signaling molecules (kinases, GTPases and adaptors) that constitute dynamic adhesion structures such as focal adhesions and podosomes .
Nevertheless, whether clustering triggers outside-in signaling to facilitate integrin activation or occurs after integrin activation is uncertain .
When the cell moves forward, it releases the integrin grip at its rear and brings it inside itself for recycling to the front, where it is then reused to make a new grip and move forward.
This type of movement is important when cancers metastasize, breaking away from the primary tumor and spreading through the rest of the body.
Cancer cells need to crawl through a tissue using integrin until they reach a blood vessel they can use to travel long distances.
Disabling the integrin mode of transport might be one method of preventing cancer from spreading.
UConn Health vascular biologists Mallika Ghosh and Linda Shapiro wondered how a common protein found in a cell’s skin, called the cell membrane, affected this type of movement.
The protein, called CD13, spikes through the cell’s membrane, with one end interacting with the inside of the cell and the other with the outside world.
CD13 has many different functions, including binding a cell in place and helping cells communicate with each other.
To test CD13’s role in cellular movement, Ghosh, Shapiro, and their colleagues first looked at mouse fibroblasts, a type of cell that makes the scaffolding that holds tissues and organs together.
They added the fibroblasts to petri dishes filled with fibronectin, a material found outside of the cell that integrin grasps. Integrin, remember, is the protein that cells use to grab on and drag themselves through a tissue. Some of the fibroblasts were normal; others had had the gene for CD13 knocked out.
The researchers found that normal fibroblasts could move through the petri dish using their integrin method with no trouble, but CD13 knock-out fibroblasts couldn’t move at all.
Then they stained the cell nucleus blue and the integrin on the cell surface green, and watched to see what happened.
The normal fibroblasts pulled all their integrin inside, and after about two hours for recycling, it reappeared on the surface.
The CD13 knock-out fibroblasts also pulled all their integrin inside after two hours, but the integrin never reappeared.
They tried the same experiment with human cervical cancer cells and got the same result.
What appeared to be happening is that CD13 acts as an organizer, gathering the freshly recycled integrin and other necessary proteins at the cell membrane so it’s ready to be pushed out when the cell needs to move.
“Without CD13, the integrins go inside and don’t come back out,” Shapiro says.
The details of how CD13 gathers the integrin in the right place involves assembly of the cell’s recycling machinery by the part of CD13 that extends inside of the cell in response to signals detected by the segment of CD13 that protrudes outside.
“And all these steps are critical for the cells to process information from the outside environment and move forward,” Ghosh says.
The researchers are now looking at different versions of integrin proteins and various binding materials such as collagen and laminin, to see if CD13 plays the same role in cell movement in tissues that use those proteins for structure.
More information: Mallika Ghosh et al, CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, β1 integrin recycling, and cell migration, Science Signaling (2019). DOI: 10.1126/scisignal.aav5938
Journal information: Science Signaling
Provided by University of Connecticut
