Abstract
Adenomatous polyposis coli (APC) is a tumor suppressor protein with important roles in Wnt signaling as
well as cytoskeletal rearrangements that govern cell migration, cell protrusion, and cell adhesion.
Because APC is a large multi‐domain protein with a wide spectrum of activities and cellular binding
partners, it has been difficult to pinpoint which of these activities and interactions are important for its
different in vivo functions. To address this, we generated a separation‐of‐function allele in APC that
specifically disrupts its actin nucleation activity, a function that until now has only been studied in vitro.
Our previous studies showed that a C‐terminal 78 kDa fragment of APC (2130‐2843), which includes the
‘basic domain’, interacts with actin monomers, nucleates actin assembly, and directly collaborates with
formins via a ‘rocket launcher’ mechanism to promote actin assembly. Here, we mapped the nucleation
activity to specific residues in the basic domain, and found that mutations at this site disrupt the ability
of APC to stimulate actin nucleation in vitro, both alone and in collaboration with formins. Importantly,
this mutation had no effect on APC’s ability to bind or bundle microtubules. In U2OS cells, silencing of
endogenous APC led to a striking loss of microtubule and mitochondria staining at the cell cortex, and a
pronounced defect in directed cell migration. Expression of an RNAi‐refractive wild‐type full‐length APC
rescued all of these defects. However, full‐length mutant APC only rescued the microtubule and
mitochondria defects, and not the cell migration defects. Thus, our results indicate that APC’s actin
nucleation activity is required for its functions in directed cell migration, but not MT capture/stability or
mitochondria transport. These findings also raise the intriguing possibility that APC‐mediated actin
nucleation facilitates the migration of colonic crypt epithelial cells, and thus contributes to its role as a
tumor suppressor.
well as cytoskeletal rearrangements that govern cell migration, cell protrusion, and cell adhesion.
Because APC is a large multi‐domain protein with a wide spectrum of activities and cellular binding
partners, it has been difficult to pinpoint which of these activities and interactions are important for its
different in vivo functions. To address this, we generated a separation‐of‐function allele in APC that
specifically disrupts its actin nucleation activity, a function that until now has only been studied in vitro.
Our previous studies showed that a C‐terminal 78 kDa fragment of APC (2130‐2843), which includes the
‘basic domain’, interacts with actin monomers, nucleates actin assembly, and directly collaborates with
formins via a ‘rocket launcher’ mechanism to promote actin assembly. Here, we mapped the nucleation
activity to specific residues in the basic domain, and found that mutations at this site disrupt the ability
of APC to stimulate actin nucleation in vitro, both alone and in collaboration with formins. Importantly,
this mutation had no effect on APC’s ability to bind or bundle microtubules. In U2OS cells, silencing of
endogenous APC led to a striking loss of microtubule and mitochondria staining at the cell cortex, and a
pronounced defect in directed cell migration. Expression of an RNAi‐refractive wild‐type full‐length APC
rescued all of these defects. However, full‐length mutant APC only rescued the microtubule and
mitochondria defects, and not the cell migration defects. Thus, our results indicate that APC’s actin
nucleation activity is required for its functions in directed cell migration, but not MT capture/stability or
mitochondria transport. These findings also raise the intriguing possibility that APC‐mediated actin
nucleation facilitates the migration of colonic crypt epithelial cells, and thus contributes to its role as a
tumor suppressor.
| Original language | English |
|---|---|
| Article number | M216 |
| Pages (from-to) | 59 |
| Journal | Molecular Biology of the Cell |
| Volume | 27 |
| Publication status | Published - 2016 |