Quitting smoking is literally the hardest thing ever. The feeling is like being nervous, starving and really needing a wee at the same time but nothing makes those feelings go away aaaaaaaaaaaaaaaaaaaaa

biochemistries
biochemistries:

Golgi Antiapoptotic proteins, or GAAPs (not to be confused with GAPs…) are a highly conserved family of membrane proteins that as their name suggests inhibit apoptosis and promote Ca2+ release from intracellular stores in the Golgi. The v-GAAP (viral GAAP) was first characterised in camelpox and vaccinia only 5 years ago, modulating virulence (as infectious agents don’t themselves undergo apoptosis).
The mitochondrion reigns supreme over apoptosis regulation, but endoplasmic reticulum (ER), lysosomes, nucleus, and Golgi apparatus can all transmit stress signals to mitochondria, and thus induce apoptosis.
Calcium ions are tasked with diverse functions in the cell, but notably in its adhesion and motility. The authors of a study in this week’s JCB hypothesise that human GAAP proteins (hGAAPs) may be implicated in these events.
Their work found that hGAAP increased store-­operated Ca2+ entry (SOCE) and thereby the activity of calpain [cysteine proteases] at newly forming protru­sions.
These hGAAP­-dependent effects regulated focal adhesion dynamics and cell migration. Indeed, inhibition or knockdown of calpain 2 abrogated the effects of hGAAP on cell spreading and migration. Our data reveal that hGAAP is a novel regulator of focal adhesion dynam­ics, cell adhesion, and migration by controlling localized Ca2+ dependent activation of calpain.
The lab used a FRET biosensor comprising CFP and YFP (Cyan and Yellow Fluorescent Proteins) to report the spatial distribution of calpain protease activity near the plasma membrane in live cells. CFP is separated from YFP by a peptide sequence that is cleaved by calpain and thus abolishing FRET (which requires the 2 to be at a short distance).
Interference reflection miscopy (IRM) was used in parallel to define the adhesion areas where cells contacted the substrate. Following the cell revealed calpain activity rose near the plasma membrane in hGAAP-overexpressing cells, and the precision of IRM measurements helped show the overlap in space and time between the increased calpain activity & focal adhesion disassembly.
In the image above, a colour scale from red (low FRET / high calpain activity) through green to blue (high FRET / non-cleaved biosensor) the cell on the right is visibly in the red at its edges, indicating high rates of protein breakdown.
Manganese ions, which like calcium are also divalent (2+), confirmed that hGAAP stimulated entry of cations across the plasma membrane thanks to its quenching of intracellular fura-2 fluorescence.
an (open access) methods video of fura-2 / Mn2+ assay is available on Jove (Journal of Visualized Experiments), complete with more background info…
The group conclude:
hGAAP enhances SOCE, which then stimulates localized activation of calpain near the plasma membrane and thereby increased cleavage of focaladhesion proteins such as FAK. Results with both small molecule inhibitors and siRNA confirm that stimulation of calpain 2 activity is required for hGAAP to promote cleavage of FAK, cell spreading and enhanced migration.The ER and Golgi usually contain relatively high concentrations of Ca2+. Targeted aequorins have shown previously that hGAAP reduces luminal Ca2+ concentrations in both the ER and Golgi. Constitutive and bidirectional cycling of membranous compartments occurs between the ER and Golgi. Depletion of luminal Ca2+ in one organelle may therefore affect the luminal Ca2+ concentration in the other.Apoptosis and survival signals are intimately linked to the ability of cells to adhere either to other cells or to the extracellular matrix. In many cases, this is orchestrated by integrin-dependent survival signals that signal via FAK. The hGAAP-dependent inhibition of apoptosis may, therefore, be directly linked with the new roles for GAAP in cell adhesion that we describe here.
Saraiva et al (2013) hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2. Journal of Cell Biology, 202: 699—713
See also:
■ English and Voeltz (2013) Endoplasmic Reticulum Structure and Interconnections with Other Organelles. Cold Spring Harbor Perspectives in Biology, 5:a013227

Life is beautiful. I love studying it

biochemistries:

Golgi Antiapoptotic proteins, or GAAPs (not to be confused with GAPs…) are a highly conserved family of membrane proteins that as their name suggests inhibit apoptosis and promote Ca2+ release from intracellular stores in the Golgi. The v-GAAP (viral GAAP) was first characterised in camelpox and vaccinia only 5 years ago, modulating virulence (as infectious agents don’t themselves undergo apoptosis).

The mitochondrion reigns supreme over apoptosis regulation, but endoplasmic reticulum (ER), lysosomes, nucleus, and Golgi apparatus can all transmit stress signals to mitochondria, and thus induce apoptosis.

Calcium ions are tasked with diverse functions in the cell, but notably in its adhesion and motility. The authors of a study in this week’s JCB hypothesise that human GAAP proteins (hGAAPs) may be implicated in these events.

Their work found that hGAAP increased store-­operated Ca2+ entry (SOCE) and thereby the activity of calpain [cysteine proteases] at newly forming protru­sions.

These hGAAP­-dependent effects regulated focal adhesion dynamics and cell migration. Indeed, inhibition or knockdown of calpain 2 abrogated the effects of hGAAP on cell spreading and migration. Our data reveal that hGAAP is a novel regulator of focal adhesion dynam­ics, cell adhesion, and migration by controlling localized Ca2+ dependent activation of calpain.

The lab used a FRET biosensor comprising CFP and YFP (Cyan and Yellow Fluorescent Proteins) to report the spatial distribution of calpain protease activity near the plasma membrane in live cells. CFP is separated from YFP by a peptide sequence that is cleaved by calpain and thus abolishing FRET (which requires the 2 to be at a short distance).

Interference reflection miscopy (IRM) was used in parallel to define the adhesion areas where cells contacted the substrate. Following the cell revealed calpain activity rose near the plasma membrane in hGAAP-overexpressing cells, and the precision of IRM measurements helped show the overlap in space and time between the increased calpain activity & focal adhesion disassembly.

In the image above, a colour scale from red (low FRET / high calpain activity) through green to blue (high FRET / non-cleaved biosensor) the cell on the right is visibly in the red at its edges, indicating high rates of protein breakdown.

Manganese ions, which like calcium are also divalent (2+), confirmed that hGAAP stimulated entry of cations across the plasma membrane thanks to its quenching of intracellular fura-2 fluorescence.

The group conclude:

hGAAP enhances SOCE, which then stimulates localized activation of calpain near the plasma membrane and thereby increased cleavage of focaladhesion proteins such as FAK. Results with both small molecule inhibitors and siRNA confirm that stimulation of calpain 2 activity is required for hGAAP to promote cleavage of FAK, cell spreading and enhanced migration.

The ER and Golgi usually contain relatively high concentrations of Ca2+. Targeted aequorins have shown previously that hGAAP reduces luminal Ca2+ concentrations in both the ER and Golgi. Constitutive and bidirectional cycling of membranous compartments occurs between the ER and Golgi. Depletion of luminal Ca2+ in one organelle may therefore affect the luminal Ca2+ concentration in the other.

Apoptosis and survival signals are intimately linked to the ability of cells to adhere either to other cells or to the extracellular matrix. In many cases, this is orchestrated by integrin-dependent survival signals that signal via FAK. The hGAAP-dependent inhibition of apoptosis may, therefore, be directly linked with the new roles for GAAP in cell adhesion that we describe here.

Saraiva et al (2013) hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2Journal of Cell Biology202: 699—713

See also:

■ English and Voeltz (2013) Endoplasmic Reticulum Structure and Interconnections with Other OrganellesCold Spring Harbor Perspectives in Biology, 5:a013227




Life is beautiful. I love studying it

lexywordsworth
Happiness is not a static state. We’ve misconceived of it completely. We think that there is some little oasis waiting where you sit around all day dabbling your feet in the pond sipping pina coladas. That is not happiness. It is getting up in the morning and not having enough time to do everything, having too many emails, and something in the day that excites you and you don’t know what’s going to happen. It’s about uncertainty, engagement, direction and motion, and it is not standing still. That is the Western problem- how we talk about the H-word. We think it is some sort of position we can achieve. It is not a position, it is a trajectory.
 Interview with Lionel Shriver in Oh Comely Magazine  (via lexywordsworth)