(Received for publication, August 25, 1994; and in revised form, October 17, 1994)
From the
Nonmuscle cell motility requires marked changes in the
consistency and shape of the peripheral cytoplasm. These changes are
regulated by a gel-sol transformation of the actin filament network,
and actin filament-severing proteins are responsible for network
solation. Macrophage Cap G, unlike all other proteins in the gelsolin
family, caps but does not sever actin filaments. Two amino acid
stretches in Cap G diverge markedly from the severing proteins: LNTLLGE and
AFHKTS. Discrete mutations in
Cap G have been generated to determine if these amino acid sequences
are critical for actin filament severing. Conversion of
LNTLLGE to the gelsolin actin-binding helix sequence (
LDDYLGG) renders Cap G capable of severing actin
filaments (half-maximal severing, 1-2 µM). Adding a
second set of mutations, converting
AFHKTS to
GFKHV, enhances severing by 10-fold (half-maximal
severing, 0.1-0.2 µM). These experiments support a
critical role for these two regions in actin filament severing and
showcase the power of gain-of-function mutations in clarifying
structure-function relationships.
Cell movement requires rapid remodeling of the peripheral cytoplasm, and remodeling is achieved by gel-sol transformations of the actin filament network. The peripheral cytoplasm of motile cells is organized into an extensive orthogonal network of actin filaments cross-linked by ABP-280. Dissolution or solation of this network is brought about by severing actin filaments. The protein thought to serve this function in many cell types is gelsolin. Understanding the mechanism by which gelsolin and other severing proteins break apart preformed filaments is critical to understanding how cells generate amoeboid movement and ingest particles by phagocytosis.
Gelsolin was
the first member of a family of actin regulatory proteins to be
described(1) . Other members include villin, adseverin,
severin, and fragmin(2) . Like gelsolin, all of these members
sever actin filaments. Macrophage Cap G, previously named
macrophage-capping protein(3, 4, 5) ,
gCap39(6) , and Mbh1(7) , is a more recently discovered
member of this family. To eliminate confusion, investigators have
agreed to rename the protein Cap G: ``Cap'' to signify that
this protein is the only member of the gelsolin family that caps the
barbed ends of actin filaments, but cannot sever them(3) , and
``G'' to emphasize that Cap G shares highest sequence
identity with gelsolin (49%
identity)(4, 6, 7) . Like severin and
fragmin, Cap G is one-half the size of gelsolin and villin, containing
three rather than six repeat domains. Cap G, like all members of the
gelsolin family, requires micromolar Ca to bind
actin.
Cap G is the only member of the gelsolin family that does not sever actin filaments. I predicted that amino acid sequences shared by all of the severing proteins in this family, but divergent in Cap G, should identify those segments critical for severing function. Indeed, two sequences in Cap G proved to be divergent. To test my hypothesis, specific mutations were made to convert these divergent stretches to the amino acid sequences found in gelsolin. A gain of function was achieved, and Cap G was converted from a strictly capping to a capping and severing protein. My investigations provide functional support for the prediction of McLaughlin et al.(8) , based on crystallographic data of segment 1 of gelsolin, that the ``actin-binding helix'' (residues 100-117 of gelsolin) plays a critical role in actin filament severing.
Figure 1:
Sequence comparisons of Cap G,
gelsolin, villin, fragmin, and severin. A, amino acid
sequences were compared within the region previously suggested to be
important for severing. The shaded areas show regions of high
identity. Two regions (in B and C) revealed deviation
of the Cap G sequence (boldfaceletters in unshadedblocks) from the consensus sequences shared
by the severing proteins. Gaps are designated by dashes. The
region of identity to the - and
-actin sequence is underlined in B. The region highly conserved among
the severing proteins, but different in Cap G, is underlined in C.
The second region in which Cap G differed from the four other proteins was located at the end of the first domain and the beginning of the second domain of gelsolin (Fig. 1C)(7) . In a highly conserved 5-amino acid sequence, GFKHV, Cap G had one amino acid insertion as well as three amino acid substitutions. While Cap G had 2 polar uncharged amino acids at the COOH-terminal end of this consensus sequence (Thr and Ser), all severing proteins were characterized by a polar charged amino acid (His) followed by a hydrophobic residue (Val).
I first investigated the functional consequences of
changing the sequence AFHKTS to
GFKHV, thus
making this amino acid stretch identical to gelsolin (Fig. 1C). As shown in Fig. 2and Fig. 5,
like native Cap G, the GFKHV mutant failed to sever preformed actin
filaments at final concentrations as high as 3 µM. The
apparent K
for actin filament capping was also
identical to that of native recombinant Cap G, 0.5 nM (data
not shown).
Figure 2: Severing activity of GFKHV mutant Cap G protein. 2 µM (final concentration) pyrenyl-actin polymerized overnight in S2 buffer containing 10 nM (final concentration) gelsolin was diluted to a final concentration of 100 nM in S2 buffer containing final concentrations of 1.5 µM (solidcircles) and 3 µM (solidsquares) GFKHV mutant Cap G. Depolymerization was monitored by a Perkin-Elmer LS5 fluorometer. No acceleration in the depolymerization was observed, consistent with a lack of severing activity.
Figure 5: Bar graph comparing the dose dependence of the initial depolymerization rates (first 2 min) for the three mutant proteins.
Next, the sequence LNTLLGE was changed to
LDDYLGG using the same methods (Fig. 1C).
The resultant mutant protein possessed calcium-sensitive severing
function ( Fig. 3and Fig. 5), although requiring high
concentrations of protein to produce significant severing (1-2
µM final concentrations). The same concentrations of
native recombinant Cap G failed to demonstrate severing activity (data
not shown). Actin filament capping of the LDDYLGG mutant was similar to
that of native Cap G (K
= 0.5 nM;
see Fig. 6).
Figure 3: Severing activity of LDDYLGG mutant Cap G. Gelsolin-pyrenyl-actin filaments prepared as described in the legend to Fig. 2were diluted to 100 nM in S2 buffer alone (opencircles) or in S2 buffer containing the LDDYLGG mutant Cap G protein (0.5 (solidcircles), 1 (solid triangles), and 2 (solidsquares) µM final concentrations). Note the concentration-dependent increase in the depolymerization rate, indicating the production of increasing numbers of uncapped filament ends due to actin filament severing.
Figure 6: Measurement of barbed-end capping by native Cap G (open squares) and by the two severing mutants LDDYLGG (closed circles) and GFKHV/LDDYLGG (closed squares). The assay is described under ``Experimental Procedures.'' Pyrenyl-actin was polymerized in S2 buffer alone or in S2 buffer containing different concentrations of Cap G or the mutant Cap G protein. Actin filaments were then diluted 40-fold in S2 buffer to a final concentration of 50 nM. The resulting final concentrations of Cap G and the mutant Cap G proteins are depicted on the right side of the graph. The depolymerization rates were slowed to a similar extent by each of the proteins, indicating similar apparent affinities for the barbed filament ends. Initial fluorescence values were corrected to allow visual comparisons of the depolymerization rates of the different samples.
A third mutant protein was also generated containing both the GFKHV and LDDYLGG mutations. As shown in Fig. 4and 5, this mutant was capable of severing actin filaments at markedly lower concentrations than the LDDYLGG mutant (one-tenth the concentration), with significant severing being observed at final concentrations between 0.1 and 0.2 µM. The capping function of this mutant was also found to be similar to that of native Cap G (Fig. 6).
Figure 4: Severing activity of GFKHV/LDDYLGG mutant Cap G. Gelsolin-pyrenyl-actin filaments were diluted in buffer alone (control (opencircles)) or in buffer containing increasing concentrations of the GFKHV/LDDYLGG mutant (0.1 (solidcircles), 0.2 (solid triangles), 0.5 (solid squares) µM final concentrations) and were monitored as described in the legend to Fig. 2. Note that approximately one-tenth the concentration used in Fig. 3was required to generate a similar degree of severing.
The severing ability of cytoplasmic gelsolin purified from rabbit alveolar macrophages was also examined using the same assay system. As shown in Fig. 7, gelsolin was capable of severing at much lower concentrations than the GFKHV/LDDYLGG mutant (approximately one-fiftieth the concentration of gelsolin produced comparable severing).
Figure 7: Severing activity of full-length gelsolin. Gelsolin-pyrenyl-actin filaments were diluted in buffer alone (control (opencircles)) or in buffer containing increasing concentrations of gelsolin (1.2 (solid circles), 5 (solid triangles), 10 (solidsquares), and 20 (open triangles) nM final concentrations) and were monitored as described in the legend to Fig. 2. Note that approximately one-fiftieth the concentration of the mutant Cap G protein used in Fig. 5was required to generate a similar degree of severing.
My finding that
the LDDYLGG region was capable of conferring severing function to a
capping protein is consistent with the three-dimensional structure of
the gelsolin-actin complex(8) . The LDDYLGG segment has been
shown to be the central region of a long -helix (extending from
residues 100 to 117 in plasma gelsolin) that interacts within the cleft
formed by actin subdomains 1 and 3. This actin-binding helix consists
of a central region of apolar side chains flanked by polar
hydrogen-bonding groups and is predicted to bring about severing by
inducing steric clashes with subdomain 2 of the adjacent actin subunit
in the same strand(8) . The present findings show that the
LDDYLGG mutation alone in Cap G can sever actin filaments only at high
concentrations, suggesting that other regions of the protein might also
play a role in severing. Conversion of a second segment from the native
sequence to that of gelsolin (i.e. addition of the GFKHV
mutation) enhances severing activity by a factor of 10. This amino acid
sequence is contained in the region of gelsolin (amino acids
150-160; see Fig. 1) known to confer severing activity to
truncated gelsolin(11) . However, the gelsolin-(1-160)
truncation mutant has recently been shown to have lower severing
activity than full-length gelsolin(9) . Similarly, although the
addition of the GFKHV mutation enhances Cap G's ability to sever,
this change fails to increase severing activity to levels observed in
full-length gelsolin. The GFKHV region in gelsolin is not required for
side binding(9) ; therefore, the mechanism by which this amino
acid sequence enhances severing in Cap G remains to be elucidated.
Significantly, none of the structural changes produced by my mutations affected Cap G's ability to cap the barbed ends of actin filaments, indicating that different structural determinants mediate the capping and severing of actin filaments. Further consideration of these gain-of-function mutant proteins is likely to divulge how other actin regulatory activities (e.g. actin nucleation, actin monomer binding, and actin filament side binding) relate to actin filament severing. Future investigations of these gain-of-function mutants promise to provide even further insight into how phagocytic cells sever actin filaments to generate the shape changes critical for amoeboid movement.