- Email: [email protected]
Functions of gelsolin: motility, signaling, apoptosis, cancer
cbb116.qxd
12/20/1999 12:23 PM
Page 103
103
Functions of gelsolin: motility, signaling, apoptosis, cancer David J Kwiatkowski Several new members of the gelsolin family have been discovered in the past year. Determination of the structure of gelsolin and identification of lysophosphatidic acid as a negative regulator provide novel functional insights. Gelsolin is an obligate downstream effector of Rac for motility in dermal fibroblasts, regulates phosphoinositide signaling pathways and ion channel function in vivo, and acts as both a regulator and effector of apoptosis. Addresses Genetics Laboratory, Hematology Division, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, MA 02115, USA; e-mail: [email protected] Current Opinion in Cell Biology 1999, 11:103–108 http://biomednet.com/elecref/0955067401100103
compact in EGTA, with the carboxy-terminal peptide overlying an actin-binding helix of domain 2, suggesting that a major structural change occurs with activation, probably also involving an extended 53 amino acid tether between domains 3 and 4, permitting the protein to straddle and sever an actin filament. Other studies also indicate that structural changes in both gelsolin and actin during severing are likely [8–11]. Inhibition of gelsolin–actin binding by phosphoinositides is probably due to competition between phosphoinositides and actin for a common binding region in the S2 domain. Remarkably, the gelsolin domain structure is identical to that of destrin and other cofilin family members despite a lack of primary sequence similarity [12]. This suggests either a single evolutionary origin for all proteins that sever or destabilize actin filaments, or convergent evolution.
© Elsevier Science Ltd ISSN 0955-0674 Abbreviations FLAP flightless I LRR associated protein LPA lysophosphatidic acid LRR leucine-rich repeat NMDA N-methyl-D-aspartate
Introduction Gelsolin is best known for its involvement in dynamic changes in the actin cytoskeleton during a variety of forms of cell motility. Gelsolin severs assembled actin filaments in two, and caps the fast-growing plus end of a free or newly severed filament (Figure 1). These interactions are regulated by Ca2+ ion (at micromolar levels) and pH (<6.5), which synergistically activate gelsolin’s binding to actin. Phosphoinositides of the D3 and D4 types release gelsolin from actin filament ends, providing sites for actin assembly. In this review I focus on recent literature (1997–1998) on gelsolin and related proteins in mammals only, and emphasize newly discovered functions of gelsolin and in vivo studies. Other more comprehensive gelsolin reviews are available in which structural aspects are emphasized [1–4].
Structure and regulation of gelsolin The primary amino acid sequence of gelsolin indicated that it consisted of six tandem copies of a core domain [5], and this same structure is present with some variation in gelsolin family members (Figure 2). Models of gelsolin structure and function based upon analyses of proteolytically-derived or bacterially-expressed fragments have been refined by determination of the crystallographic structure of domain 1 in complex with actin [6], and that of fulllength gelsolin in submicromolar free Ca2+ [7••]. As expected, the conserved residues in each of the six domains define the basic domain structure, whereas actin binding and other functions involve residues that vary between the domains. The conformation of the protein is
Gelsolin family members and expression A new low abundance cytoplasmic gelsolin isoform, gelsolin-3, that differs from cytoplasmic gelsolin only by an additional 11 amino acid residues at its amino-terminus, has been identified [13]. Adseverin (scinderin) is the closest homologue to gelsolin and was originally discovered in bovine adrenal gland (Figure 2); however, in murine and human adult tissues, adseverin expression is highest in kidney and gut (>0.1% total cell protein in certain renal tubular epithelial cells), with lower levels in the thymus and adrenal gland (but not medulla), and very low to no expression elsewhere [14•]. Adseverin is also highly and variably expressed in hematopoietic lineage cells, where a novel isoform missing domain 5 (see Figure 2) was found [15•], and in endochondral bone primordia (Arai M, Kwiatkowski DJ, unpublished data). These observations conflict with the proposed models of a function for adseverin in exocytosis, at least in the majority of murine and human tissues [16]; moreover, the expression pattern of capG, adseverin and gelsolin is highly complementary in the mouse [14•] (Arai M, Kwiatkowski DJ, unpublished data), suggesting that the distinct functional characteristics of these proteins lead to distinct patterns of cellular expression according to the motile capacity of the cell. Human flightless, a gelsolin homologue with an amino-terminal leucine-rich repeat (LRR) (Figure 2) has been shown to bind to actin, and to bind to a newly described protein — flightless I LRR associated protein (FLAP) of size 626 amino acids — through its LRR [17•]. Advillin is a gelsolin family member that is most closely related to villin (59% amino-acid identity), including a headpiece domain [18•]. It is highly expressed in dorsal root and trigeminal ganglia during embryonic development and
cbb116.qxd
12/20/1999 12:23 PM
104
Page 104
Cytoskeleton
Figure 1
Ca2+/H+ Gelsolin Severing & capping
Nucleation & capping Uncapping
p
p p
A model of gelsolin’s interaction with actin in the cell. At rest, gelsolin is inactive, and depicted as a compact six domain structure. In response to micromolar Ca2+ or pH < 6.5, gelsolin opens up to become active in either severing or binding to actin monomers, resulting in a capped actin filament. In response to clustered phosphoinositides (zigzag-tailed hexagons) and possibly lysophosphatidic acid, gelsolin uncaps from the filament, and resumes a closed configuration in low Ca2+ and normal pH.
p
p
D3 and D4 phosphoinositides, ? lysophosphatidic acid Current Opinion in Cell Biology
at lower levels in adult uterine and intestinal epithelial cells. Advillin expression is induced in murine embryo fibroblasts in response to tunicamycin treatment and possibly other forms of cellular stress [19]. Supervillin (205 kDa) is another newly identified gelsolin family member that participates in interactions between actin filaments and membrane [20•]. It consists of an amino-terminal half which has nuclear targeting signals, and a carboxy-terminal half which in general has weak similarity to villin, but also contains more conserved regions corresponding to F-actin binding regions (Figure 2).
Gelsolin-related amyloidosis pathogenesis Recent evidence that gelsolin binds lysophosphatidic acid (LPA) [21••], which is present in diverse extracellular sites, suggests that an important function of secreted gelsolin is as a lipid carrier protein in addition to its postulated role in actin clearance [22]. Gelsolin-related amyloidosis is a rare hereditary amyloid polyneuropathy which is a result of Asp187→Asn or Asp187→Try mutations in gelsolin. These mutations cause an aberrant intracellular cleavage of secreted gelsolin between residues 172 and 173 leading to production of the amyloidogenic 173–243 fragment [23]. The 172/173 cleavage appears to be due to disruption of the Cys188–Cys201 disulfide bond in plasma gelsolin and to occur at highest frequency in neuronal cells, consistent with the pattern of organ involvement in this disease [24•].
The gelsolin-null mouse Gelsolin-null mice express no cytoplasmic or plasma gelsolin, and in mixed-strain backgrounds are completely viable with normal longevity under animal colony conditions [25]. They have proven to be a critical resource in the
analysis of gelsolin function in vivo [26••–28••,29•]. When the gelsolin gene defect is bred into either BALB/c or C57/black backgrounds, nearly all gelsolin-null pups die between embryonic day 17 and postnatal day 21 (Witke W, Kwiatkowski DJ, unpublished data), indicating that in concert with the multiple gene defects present in these strains, gelsolin is necessary for survival.
Gelsolin’s role in motility Analysis of dermal fibroblasts from the gelsolin-null mice has strengthened the view that gelsolin has a critical role in actin dynamics [26••]. The gelsolin-null cells display reduced ruffling activity and translocational motility in response to multiple stimuli and, in multiple assay systems, have increased amounts of F-actin in stress fibers and crawl by a distinctive pseudopod-like extension process. Both cultured cells and tissues from the gelsolinnull animals display a fivefold increase in the expression of the Rac GTPase, and the motility defect and Rac overexpression are repaired by gelsolin transfection. Filopodial protrusion is normal in these cells, suggesting that gelsolin serves a critical ‘effector’ function specific for cytokine motility signals transmitted by the Rac GTPase to the actin architecture (Figure 3). Interestingly, this effect is not seen in gelsolin-null embryo fibroblasts, consistent with different motility mechanisms during embryogenesis (Azuma T, Kwiatkowski DJ, unpublished data). Although gelsolin did not appear to be important in a cellfree system examining Listeria motility [30], gelsolin-transfected NIH3T3 cell lines displayed a proportional increase in Listeria motility [31•], consistent with a complex effect of gelsolin expression not amenable to cellfree analysis. The importance of gelsolin activity for motility has also been seen in gingival fibroblasts [32], and
cbb116.qxd
12/20/1999 12:23 PM
Page 105
Functions of gelsolin: motility, signaling, apoptosis, cancer Kwiatkowski
in epidermal growth factor receptor mediated motility in NR6 fibroblasts [33].
Figure 2 1
Signaling from Rac to gelsolin was also discovered in neutrophil extracts [34 •]. Release of actin from 1:1 gelsolin–actin complexes occurred independently of phosphatidylinositol 3-kinase and cellular phosphoinositides, and could be produced by GTP-loaded Rac or GTPloaded heterotrimeric G proteins. These observations contrast with models of gelsolin regulation derived from platelet studies [35], and may reflect the differing cell types and/or cell preparation methods. Movement of gelsolin to the Triton-X-100-insoluble cytoskeleton was observed during neutrophil adherence to fibronectin-coated plastic dishes [36].
2
3
4
5
6 gelsolin adseverin adseverin (D5) capG
LRR
flightless villin advillin
NT
Cultured embryonic hippocampal neurons were used to explore the function of gelsolin during neurite growth [29•]. Although ruffling activity and motility of growth cones were not affected, gelsolin-null neurites had an increased number of filopodia along their length. Filopodial extension was normal, but retraction was delayed, suggesting that gelsolin functioned to degrade the actin filament core during filopodial retraction.
Ion-channel regulation Modulation of ion-channel function by the actin cytoskeleton is increasingly understood [37,38], and the role of gelsolin in this process has now been shown in vivo [27••]. Gelsolin-null embryonic hippocampal neurons have increased filamentous actin, enhanced NMDA receptor-mediated currents, and enhanced Ca2+ intake and cell death in response to glutamate stimulation in comparison to wild-type neurons; moreover, seizureinduced damage to hippocampal neurons [27••] and ischemia-induced damage to the cortex (Endres M, Moscowitz MA, Kwiatkowski DJ, unpublished data) are both enhanced in gelsolin-null mice compared to wildtype animals.
Signaling and lipids Although originally viewed in the context of regulation of gelsolin function, the phosphoinositide–gelsolin interaction is now also recognized to modulate lipid signaling events. Gelsolin and capG binding to phosphoinositides is significantly enhanced by micromolar Ca2+ and also increased by mild acidification [39•]. The ability of gelsolin but not profilin to alter the structure of phosphatidylinositol 4,5-bisphosphate aggregates also reflects the high affinity of gelsolin for phosphoinositides [40•]; moreover, gelsolin and capG overexpression in NIH3T3 cells has significant effects on signaling through cytokine-stimulated pathways activating either phospholipase Cβ for phospholipase Cγ [41••]. Gelsolin has also been isolated in complexes with an increasing array of lipases and kinases, including phospholipases Cγ1, Cδ, and D, phosphatidylinositol 3-kinase, and
105
supervillin HP Current Opinion in Cell biology
Eight members of the gelsolin family of proteins (mammals only) are modeled. The core domain which is repeated six times is shown. LRR, leucine-rich repeat; HP, headpiece; NT, nuclear translocation domain.
c-src [42–44,45•,46•]. These results also suggest that gelsolin may alter lipid signaling pathways, either through direct binding to these proteins or, more likely (in my opinion), through joint binding to clustered phosphoinositides. Phosphatidylinositol 4,5-bisphosphate also enhances phosphorylation of gelsolin by the Src kinase to a maximum level of 0.18 mol phosphate per mol gelsolin in vitro [47], but there is no evidence for gelsolin phosphorylation in vivo. In osteoclasts, osteopontin stimulation induces formation of a signaling complex including gelsolin, phosphatidylinositol 3-kinase, and c-src, that is required for bone resorption [46•]. Gelsolin and other family members bind to LPA with high affinity [21••]. This interaction requires somewhat higher LPA concentrations than those needed for phosphatidylinositol 4,5-bisphosphate binding, but LPA also blocks gelsolin’s severing activity and uncaps gelsolin from filament ends. LPA acts cooperatively with phosphoinositides in inhibiting gelsolin’s interaction with actin and this provides another means for the regulation of gelsolin activity in cells.
Apoptosis Caspase-3 is a core effector protease in a protease cascade that is activated during apoptosis [48]. Gelsolin is a prominent substrate of caspase-3 in vitro [28••,49•], is cleaved during apoptosis in multiple cell types, and the amino-terminal cleavage product (residues 1–352) has Ca 2+ unregulated actin filament severing activity [28••,50]. Expression of this fragment in cells leads to apoptosis and gelsolin-null neutrophils progress to apoptotic cell death more slowly than wild-type neutrophils, consistent with an important role for the gelsolin
cbb116.qxd
12/20/1999 12:23 PM
106
Page 106
Cytoskeleton
Figure 3
Bradykinin
Cdc42
EGF PDGF Insulin
LPA
Rac
Rho Gelsolin
Filopodia
Ruffling Lamellipodia
Abundant actin stress fibers
family with distinctive expression patterns, domain structure and functions [13,15•,18•,20•]. Further insight into the mechanism of severing awaits the determination of gelsolin structure in activating conditions [7••]. Fibroblast and neutrophil studies indicate a signaling pathway from the Rac GTPase to gelsolin [26••,34•]. Gelsolin has unexpected roles in neurite filopodial retraction, neuronal ion channel modulation, and as a regulator and mediator of apoptosis [27 ••,28 ••, 29•,50,51••]. It also regulates phosphoinositide signaling [41••], and is regulated by and may modulate signaling by LPA [21••]. Gelsolin appears to be important in carcinogenesis [2,56••], but the mechanism is uncertain and could relate to its effects on motility, Rac or lipid signaling, and/or apoptosis.
Current Opinion in Cell Biology
Note added in proof A model for motility signaling within dermal fibroblasts. Cytokines signal for motility via three Rho family GTPases with differing effects. Gelsolin is an obligate effector of Rac-mediated motility and ruffling activity. In gelsolin-null cells, cytoplasmic sensing in some manner leads to upregulation of Rac expression which compensates somewhat but does not lead to normal motility.
The data referred to as Endres M, Moscowitz MA, Kwiatkowski DJ, unpublished data has now been published [57•].
Acknowledgements Supported by National Institutes of Health grants HL48743 and HL54188.
fragment in mediating apoptotic progression. Gelsolin, however, has also been found to inhibit apoptosis following several stimuli in Jurkat cells (a human T-cell line), when expressed at three to seven times normal levels [51••]. One possible resolution of these conflicting observations is that gelsolin, in complex with intracellular components such as phosphoinositides, might serve as a competitive inhibitor of caspase-3 (Azuma T, Kwiatkowski DJ, unpublished data).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Liu Y-T, Rozelle AL, Yin HL: The gelsolin family of actin filament severers and cappers. In G proteins, Cytoskeleton and Cancer. Edited by Maruta HK, Kohama K. Austin TX: RG Landes Company; 1998:19-35.
2.
Kuzumaki N, Fujita H, Tanaka M, Sakai N, Ohtsu M: Tumor suppressive function of gelsolin. In G Proteins, Cytoskeleton and Cancer. Edited by Maruta HK, Kohama K. Austin, TX: RG Landes Company; 1998:121-131.
3.
McGough A: F-actin-binding proteins. Curr Opin Struct Biol 1998, 8:166-176.
4.
Janmey PA, Stossel TP, Allen PG: Deconstructing gelsolin: identifying sites that mimic or alter binding to actin and phosphoinositides. Chem Biol 1998, 5:R81-R85.
5.
Kwiatkowski DJ, Stossel TP, Orkin SH, Mole JE, Colten HR, Yin HL: Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature 1986, 323:455-458.
6.
McLaughlin P, Gooch J, Mannherz H-G, Weeds A: Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature 1993, 364:685-692.
Cancer Gelsolin expression is downregulated in 60–90% of tumors during carcinogenesis [2] of the breast [52], colon, stomach, bladder [2], prostate [53], and lung [54•]. In addition, a Pro321→His mutation in gelsolin was identified as a suppressor of EJ-ras-mediated transformation of NIH3T3 cells: Pro321→His gelsolin and wild-type gelsolin inhibit the growth of ras-transformed NIH3T3 and a bladder cancer cell line, respectively, in the nude mouse [2]. Gelsolin expression is also increased by treatment of cells with either trichostatin A or radicicol, which have growth-suppressing, apoptosis-inducing activities [55•]. We have observed, however, that high level expression of gelsolin in early stage non-small-cell lung cancer occurs in 14% of patients, and provides highly significant negative prognostic information [56••]. In addition, the gelsolin-null mice show no increased incidence of tumors (Witke W, Kwiatkowski DJ, unpublished data).
7. ••
Burtnick LD, Koepf EK, Grimes J, Jones EY, Stuart DI, McLaughlin PJ, Robinson RC: The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation. Cell 1997, 90:661-670. The crystal structure of gelsolin in EGTA is presented. The modular domain structure of the protein is confirmed, and connector peptides identified. The structure is compact and a model for how it opens during Ca2+ activation is presented, although the structure in Ca2+ is not currently available. Residues that bind phosphoinositides are seen adjacent to those which bind to the side of an actin filament, explaining phosphoinositide-mediated uncapping and inhibition of severing. 8.
McGough A, Chiu W, Way M: Determination of the gelsolin binding site on F-actin: implications for severing and capping. Biophys J 1998, 74:764-772.
9.
Khaitlina S, Hinssen H: Conformational changes in actin induced by its interaction with gelsolin. Biophys J 1997, 73:929-937.
Conclusion Gelsolin-3, adseverin (D5 isoform), advillin, and supervillin are newly isolated members of the gelsolin
cbb116.qxd
12/20/1999 12:23 PM
Page 107
Functions of gelsolin: motility, signaling, apoptosis, cancer Kwiatkowski
10. Pope BJ, Gooch JT, Weeds AG: Probing the effects of calcium on gelsolin. Biochemistry 1997, 36:15848-15855. 11. Feinberg J, Kwiatek O, Astier C, Diennet S, Mery J, Heitz F, Benyamin Y, Roustan C: Capping and dynamic relation between domains 1 and 2 of gelsolin. J Pept Sci 1998, 4:116-127. 12. Hatanaka H, Ogura K, Moriyama K, Ichikawa S, Yahara I, Inagaki F: Tertiary structure of destrin and structural similarity between two actin-regulating protein families. Cell 1996, 85:1047-1055. 13. Vouyiouklis DA, Brophy PJ: A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system. J Neurochem 1997, 69:995-1005. 14. Lueck A, Brown D, Kwiatkowski DJ: The actin-binding proteins • adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine. J Cell Sci 1998, 111:3633-3643. In the mouse and humans, adseverin is found to be most highly expressed in certain renal tubular and intestinal epithelial cells, with lower levels in the thymus, adrenal gland (but not medulla), and uterus, and very low to no expression elsewhere. In addition, expression of adseverin and gelsolin in the kidney are complementary, occurring for the most part in different tubular epithelial cell populations. 15. Robbens J, Louahed J, De Pestel K, Van Colen I, Ampe C, • Vandekerckhove J, Renauld JC: Murine adseverin (D5), a novel member of the gelsolin family, and murine adseverin are induced by interleukin-9 in T-helper lymphocytes. Mol Cell Biol 1998, 18:4589-4596. In the mouse, adseverin was found to be widely expressed but at low levels, with higher amounts in hematopoietic cells, and significant induction during cytokine treatment in some cell populations. The D5 isoform, which is expressed only in hematopoietic cells, lacks the fifth and part of the sixth domain, but has normal Ca2+ activated severing activity, but no nucleating activity. 16. Zhang L, Marcu MG, Nau-Staudt K, Trifaro JM: Recombinant scinderin enhances exocytosis, an effect blocked by two scinderin-derived actin-binding peptides and PIP2. Neuron 1996, 17:287-296. 17. •
Liu YT, Yin HL: Identification of the binding partners for flightless I, a novel protein bridging the leucine-rich repeat and the gelsolin superfamilies. J Biol Chem 1998, 273:7920-7927. Human flightless I binds to actin–sepharose. The yeast two-hybrid method was used to identify flightless I LRR associated protein (FLAP), a binding partner of the flightless I LRR. FLAP is widely expressed and consists of 626 amino acids, but has unknown function. 18. Marks PW, Arai M, Bandura JL, Kwiatkowski DJ: Advillin (p92): a new • member of the gelsolin/villin family of actin regulatory proteins. J Cell Sci 1998, 111:2129-2136. A new member of the gelsolin family is described that is most closely homologous to villin. Advillin shares the six domain structure of other gelsolin family members and, in addition, has a carboxy-terminal headpiece domain that is similar to the headpiece domain of villin. Advillin is expressed at highest levels in developing and adult ganglia in the mouse, with much lower levels expressed in the uterus and intestine, and little to none elsewhere. 19. Wang XZK, Kuroda M, Sok J, Batchvarova N, Kimmel R, Chung P, Zinszner H, Ron D: Identification of novel stress-induced genes downstream of chop. EMBO J 1998, 17:3619-3630. 20. Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ: Supervillin • (p205): a novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily. J Cell Biol 1997, 139:1255-1269. Supervillin is purified and cloned. It is a 205 kDa protein that purifies with neutrophil plasma membranes and binds to the sides of actin filaments in overlay assays. The protein is found in different intracellular locations in neutrophils, HeLa and MDCK cells, depending upon culture conditions and treatments. Structurally it consists of an amino-terminal half with apparent nuclear localization function, and a carboxy-terminal half with weak overall similarity to villin, and stronger similarity in regions of actin-binding activity.
107
23. Maury CP, Sletten K, Totty N, Kangas H, Liljestrom M: Identification of the circulating amyloid precursor and other gelsolin metabolites in patients with G654A mutation in the gelsolin gene (Finnish familial amyloidosis): pathogenetic and diagnostic implications. Lab Invest 1997, 77:299-304. 24. Paunio T, Kangas H, Heinonen O, Buc-Caron MH, Robert JJ, • Kaasinen S, Julkunen I, Mallet J, Peltonen L: Cells of the neuronal lineage play a major role in the generation of amyloid precursor fragments in gelsolin-related amyloidosis. J Biol Chem 1998, 273:16319-16324. The authors disrupt the Cys188–Cys201 disulfide bond in gelsolin by sitedirected mutagenesis and find that aberrant intracellular processing occurs, similar to that seen in Asp187→Asn amyloidogenic gelsolin. The implication is that the Asp187→Asn mutation disrupts this disulfide bond, leading to cleavage. In addition, this intracellular cleavage occurs at a higher rate in neuronal lineage cells than in other cell types. 25. Witke W, Sharpe AH, Hartwig JH, Azuma T, Stossel TP, Kwiatkowski DJ: Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell 1995, 81:41-51. 26. Azuma T, Witke W, Stossel TP, Hartwig JH, Kwiatkowski DJ: Gelsolin •• is a downstream effector of Rac for fibroblast motility. EMBO J 1998, 17:1362-1370. Gelsolin-null dermal fibroblasts are characterized in detail in comparison to wild-type fibroblasts. The gelsolin-null cells have increased F-actin, and reduced motility and pinocytosis in several assay systems. They crawl using a distinctive filopodia-like structure in contrast to the ruffles and lamellipodia present in the wild-type cells. Both the gelsolin-null cells and tissues from the gelsolin-null mice show expression of Rac at fivefold higher levels than wild-type cells, and the motility defect and Rac overexpression are reverted to wild-type by transfection of gelsolin. The implication is that gelsolin is an obligate element downstream of the Rac GTPase for motility. 27. ••
Furukawa K, Fu W, Li Y, Witke W, Kwiatkowski DJ, Mattson MP: The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J Neurosci 1997, 17:8178-8186. Primary hippocampal gelsolin-null neurons have decreased actin filament depolymerization and increased Ca2+ influx in response to glutamate compared to wild-type neurons. Enhanced currents in response to NMDA were also seen in patch-clamp experiments, as a result of a lack of current rundown. Cultured neurons showed greater cell death in response to high-dose glutamate, and seizure-induced damage to hippocampal pyramidal neurons was increased in adult gelsolin-null mice. The implication is that gelsolin has an important role in modulating neuronal ion channel function in response to excitotoxicity in vivo. 28. Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, •• McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ, Williams LT: Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 1997, 278:294-298. Screening of 100,000 murine embryo fibroblast cDNA clones in an in vitro transcription-translation reaction led to identification of gelsolin as the most prominent substrate of caspase-3. The amino-terminal gelsolin fragment generated by caspase-3 cleavage has unregulated actin filament severing activity, and when expressed in cells causes apoptosis. Gelsolin-null neutrophils have a slower rate of apoptotic death in response to tumor necrosis factor induction, compared to wild-type neutrophils. The implication is that gelsolin has an important role in apoptotic progression. 29. Lu M, Witke W, Kwiatkowski DJ, Kosik KS: Delayed retraction of • filopodia in gelsolin null mice. J Cell Biol 1997, 138:1279-1287. Although growth cone extension, ruffling activity, and translocational rates are identical in cultured gelsolin null neurons compared to wild-type, the null neurons have increased numbers of neurite filopodia. This is due to delayed filopodial retraction that implicates a function of gelsolin in dissassembling the F-actin rich core of the filopodia. 30. Rosenblatt J, Agnew BJ, Abe H, Bamburg JR, Mitchison TJ: Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails [see comments]. J Cell Biol 1997, 136:1323-1332.
21. Meerschaert K, De Corte V, De Ville Y, Vandekerckhove J, •• Gettemans J: Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid. EMBO J 1998, 17:5923-5932. An extensive survey of phospholipids led to the discovery that gelsolin binds to lysophosphatidic acid (LPA). LPA binding affects gelsolin function in a manner similar to phosphoinositides, but requires significantly higher concentrations of LPA. LPA has synergistic effects on gelsolin function when added to phosphoinositides; therefore, LPA may act as another intracellular component that regulates gelsolin activity on the actin cytoskeleton. Secreted gelsolin may also serve as a carrier of LPA and perhaps other phospholipids, significantly affecting their presentation to and function on cells.
31. Laine RO, Phaneuf KL, Cunningham CC, Kwiatkowski D, Azuma T, • Southwick FS: Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells. Infect Immun 1998, 66:3775-3782. Listeria motility and actin tail lengths were both significantly increased in NIH3T3 cells that had been transfected to overexpress gelsolin, and the increases were proportional to the level of gelsolin expression.
22. Suhler E, Lin W, Yin HL, Lee WM: Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock, and myonecrosis. Crit Care Med 1997, 25:594-598.
32. Arora PD, McCulloch CAG: Dependence of fibroblast migration on actin severing activity of gelsolin. J Biol Chem 1996, 271:20516-20523.
cbb116.qxd
12/20/1999 12:23 PM
108
Page 108
Cytoskeleton
33. Chen P, Murphy-Ullrich JE, Wells A: A role for gelsolin in actuating epidermal growth factor receptor-mediated cell motility. J Cell Biol 1996, 134:689-698.
oligonucleotides against c-Src led to reduced c-Src expression, reduced association between gelsolin and c-Src in osteoclast extracts, reduced associated phosphatidylinositol 3-kinase activity and reduced bone resorption.
34. Arcaro A: The small GTP-binding protein Rac promotes the • dissociation of gelsolin from actin filaments in neutrophils. J Biol Chem 1998, 273:805-813. Gelsolin–actin complexes are present in resting neutrophils and are dissociated by fMLP (f-Met-Leu-Phe, a chemotactic peptide for neutrophils) activation. Here this effect is shown to be insensitive to wortmannin or kinase inhibitor treatment. In a cell-free system, gelsolin–actin dissociation was achieved by addition of GTP-loaded Rac or heterotrimeric G proteins, implicating a signaling pathway between Rac and gelsolin that is independent of phosphoinositides.
47.
35. Barkalow K, Witke W, Kwiatkowski DJ, Hartwig JH: Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein. J Cell Biol 1996, 134:389-399. 36. Wang JS, Coburn JP, Tauber AI, Zaner KS: Role of gelsolin in actin depolymerization of adherent human neutrophils. Mol Biol Cell 1997, 8:121-128. 37.
Cantiello H: Role of actin filament organization in cell volume and ion channel regulation. J Exp Zool 1997, 279:425-435.
38. Lascola CD, Nelson DJ, Kraig RP: Cytoskeletal actin gates a Clchannel in neocortical astrocytes. J Neurosci 1998, 18:1679-1692. 39. Lin KM, Wenegieme E, Lu PJ, Chen CS, Yin HL: Gelsolin binding to • phosphatidylinositol 4,5-bisphosphate is modulated by calcium and pH. J Biol Chem 1997, 272:20443-20450. Superdex gel filtration was used as a primary assay to demonstrate that micromolar Ca2+ increases the affinity of gelsolin (eightfold) and capG (fourfold) for phosphatidylinositol 4,5-bisphosphate. Lowering the pH from 7.5 to 7.0 reduced the level of Ca2+ required for this effect. The implication is that during cell activation, the affinity of gelsolin and capG for phosphoinositides is increased, potentially altering phosphoinositides signaling pathways. 40. Flanagan LA, Cunningham CC, Chen J, Prestwich GD, Kosik KS, • Janmey PA: The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and tau. Biophys J 1997, 73:1440-1447. Divalent cations cause phosphatidylinositol 4,5-bisphosphate to aggregate into clusters of striated filaments. Gelsolin and tau, but not profilin, affected the structure of these aggregates, confirming gelsolin’s high affinity binding and suggesting it can alter phosphatidylinositol 4,5-bisphosphate distribution in the cell membrane. 41. Sun H, Lin K, Yin HL: Gelsolin modulates phospholipase C activity •• in vivo through phospholipid binding. J Cell Biol 1997, 138:811-820. Utilizing NIH3T3 cells stably transfected to overexpress gelsolin or capG, it is shown that overexpression of either protein strongly inhibits bradykininstimulated phospholipase Cβ and weakly inhibits tyrosine kinase-stimulated phospholipase Cγ. Specificity of the gelsolin effect on phospholipase Cβ was confirmed in washout and addback experiments performed on permeabilized cells. The implication is that these proteins can significantly affect phosphoinositide-signaling pathways in cells. 42. Banno Y, Nakashima T, Kumada T, Ebisawa K, Nonomura Y, Nozawa Y: Effects of gelsolin on human platelet cytosolic phosphoinositidephospholipase C isozymes. J Biol Chem 1992, 267:6488-6494. 43. Steed PM, Nagar S, Wennogle LP: Phospholipase D regulation by a physical interaction with the actin- binding protein gelsolin. Biochemistry 1996, 35:5229-5237. 44. Singh SS, Chauhan A, Murakami N, Chauhan VP: Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity. Biochemistry 1996, 35:16544-16549. 45. Baldassare JJ, Henderson PA, Tarver A, Fisher GJ: Thrombin • activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cγγ1. Biochem J 1997, 324:283-287. Co-immunoprecipitation experiments demonstrate that phospholipase Cγ is associated with a gelsolin–actin complex in fresh unstimulated platelets, and that this three-protein complex dissolves in response to thrombin stimulation, requiring platelet aggregation and tyrosine phosphorylation events. Phospholipase Cγ activity increases concurrent with release from gelsolin. 46. Chellaiah M, Fitzgerald C, Alvarez U, Hruska K: c-Src is required for • stimulation of gelsolin-associated phosphatidylinositol 3-kinase. J Biol Chem 1998, 273:11908-11916. Osteopontin stimulation of osteoclasts induces binding of c-Src to gelsolin in co-immunoprecipitation assays, in addition to phosphatidylinositol 3-kinase (as shown previously by these authors). Treatment of osteoclasts with antisense
De Corte V, Gettemans J, Vandekerckhove J: Phosphatidylinositol 4,5-bisphosphate specifically stimulates PP60(c- src) catalyzed phosphorylation of gelsolin and related actin-binding proteins. FEBS Lett 1997, 401:191-196.
48. Cryns V, Yuan J: Proteases to die for. Genes Dev 1998, 12:1551-1570. 49. Kamada S, Kusano H, Fujita H, Ohtsu M, Koya RC, Kuzumaki N, • Tsujimoto Y: A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA 1998, 95:8532-8537. A mutant caspase-3 was used in the yeast two-hybrid system to identify binding partners of caspase-3. Gelsolin was identified as a prominent substrate, and its cleavage confirmed in vitro. 50. Geng Y-JA T, Tang JX, Hartwig JH, Muszynski M, Libby P, Kwiatkowski DJ: Caspase-3 induced gelsolin fragmentation contributes to actin cytoskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle cells exposed to proinflammatory cytokines. Eur J Cell Biol 1999, 50:294-302. 51. Ohtsu M, Sakai N, Fujita H, Kashiwagi M, Gasa S, Shimizu S, •• Eguchi Y, Tsujimoto Y, Sakiyama Y, Kobayashi K, Kuzumaki N: Inhibition of apoptosis by the actin-regulatory protein gelsolin. EMBO J 1997, 16:4650-4656. Stable gelsolin-overexpressing transfectant lines were derived from the Jurkat cell line, a human T-cell line. These cell lines were found to have marked resistance to apoptotic induction to anti-fas antibodies, C2-ceramide, and dexamethasone, and a marked reduction in the production of caspase-3 activity. The implication is that gelsolin is blocking apoptosis at some critical and universal early step in these cells. 52. Asch HL, Head K, Dong Y, Natoli F, Winston JS, Connolly JL, Asch BB: Widespread loss of gelsolin in breast cancers of humans, mice, and rats. Cancer Res 1996, 56:4841-4845. 53. Prasad SC, Thraves PJ, Dritschilo A, Kuettel MR: Protein expression changes associated with radiation-induced neoplastic progression of human prostate epithelial cells. Electrophoresis 1997, 18:629-637. 54. Dosaka-Akita H, Hommura F, Fujita H, Kinoshita I, Nishi M, • Morikawa T, Katoh H, Kawakami Y, Kuzumaki N: Frequent loss of gelsolin expression in non-small cell lung cancers of heavy smokers. Cancer Res 1998, 58:322-327. Gelsolin expression was reduced in twelve out of twelve human lung cancer cell lines — markedly so (<10% normal levels) in ten of the twelve. Of 88 primary resected tumors 48 (55%) had no gelsolin expression, and there was no correlation with histologic type or stage. 55. Kwon HJ, Yoshida M, Nagaoka R, Obinata T, Beppu T, Horinouchi S: • Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression. Oncogene 1997, 15:2625-2631. Radicicol, an inhibitor of src-family kinases, reverts the morphology of transformed fibroblasts to untransformed fibroblasts. In two-dimensional gel analyses, gelsolin was identified as being prominently upregulated in radicicol-treated cells, similar to what the authors have seen previously with trichostatin A treatment. Injection of anti-gelsolin antibodies inhibited the morphologic reversion induced by radicicol in T24 and HeLa cells. 56. Shieh DGJ, Sugarbaker DJ, Herndon J, Kwiatkowski DJ: Motility as a •• prognostic factor in non-small cell lung cancer: role of gelsolin expression. Cancer 1999, 85:47-57. Analysis of histologic sections from 229 patients with stage I non-small cell lung cancer was performed. Rac and ABP-280 expression (an actin filament crosslinking protein, also known as filamin) appeared to have no influence on prognosis in a pilot study, but gelsolin expression was important. The 32 patients who expressed gelsolin highly (either uniformly or focally in their tumors) had a fourfold higher relative risk of cancer recurrence, and gelsolin expression was a greater risk factor than any other variable examined. 57. •
Endres M, Fink K, Zhu J, Stagliano NE, Bondala V, Geddes JW, Azuma T, Mattson MP, Kwiatkowski DJ, Moscowitz MA: Neuroprotective effects of gelsolin and cytochalasin D during murine stroke. J Clin Invest 1999, in press. Cultured gelsolin-null neurons have enhanced cell death and sustained elevation of Ca2+ levels after glucose/oxygen deprivation. In addition, infarct volumes are 36% greater in gelsolin-null mice than wild-type controls after transient middle cerebral artery occlusion. This increased stroke volume is eliminated by intraventricular injection of cytochalasin D, which also decreases stroke volume in wild-type mice. The implication is that modulation of the actin filament architecture by gelsolin and analogues such as cytochalasin D may improve the outcome from stroke.
12/20/1999 12:23 PM
Page 103
103
Functions of gelsolin: motility, signaling, apoptosis, cancer David J Kwiatkowski Several new members of the gelsolin family have been discovered in the past year. Determination of the structure of gelsolin and identification of lysophosphatidic acid as a negative regulator provide novel functional insights. Gelsolin is an obligate downstream effector of Rac for motility in dermal fibroblasts, regulates phosphoinositide signaling pathways and ion channel function in vivo, and acts as both a regulator and effector of apoptosis. Addresses Genetics Laboratory, Hematology Division, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, MA 02115, USA; e-mail: [email protected] Current Opinion in Cell Biology 1999, 11:103–108 http://biomednet.com/elecref/0955067401100103
compact in EGTA, with the carboxy-terminal peptide overlying an actin-binding helix of domain 2, suggesting that a major structural change occurs with activation, probably also involving an extended 53 amino acid tether between domains 3 and 4, permitting the protein to straddle and sever an actin filament. Other studies also indicate that structural changes in both gelsolin and actin during severing are likely [8–11]. Inhibition of gelsolin–actin binding by phosphoinositides is probably due to competition between phosphoinositides and actin for a common binding region in the S2 domain. Remarkably, the gelsolin domain structure is identical to that of destrin and other cofilin family members despite a lack of primary sequence similarity [12]. This suggests either a single evolutionary origin for all proteins that sever or destabilize actin filaments, or convergent evolution.
© Elsevier Science Ltd ISSN 0955-0674 Abbreviations FLAP flightless I LRR associated protein LPA lysophosphatidic acid LRR leucine-rich repeat NMDA N-methyl-D-aspartate
Introduction Gelsolin is best known for its involvement in dynamic changes in the actin cytoskeleton during a variety of forms of cell motility. Gelsolin severs assembled actin filaments in two, and caps the fast-growing plus end of a free or newly severed filament (Figure 1). These interactions are regulated by Ca2+ ion (at micromolar levels) and pH (<6.5), which synergistically activate gelsolin’s binding to actin. Phosphoinositides of the D3 and D4 types release gelsolin from actin filament ends, providing sites for actin assembly. In this review I focus on recent literature (1997–1998) on gelsolin and related proteins in mammals only, and emphasize newly discovered functions of gelsolin and in vivo studies. Other more comprehensive gelsolin reviews are available in which structural aspects are emphasized [1–4].
Structure and regulation of gelsolin The primary amino acid sequence of gelsolin indicated that it consisted of six tandem copies of a core domain [5], and this same structure is present with some variation in gelsolin family members (Figure 2). Models of gelsolin structure and function based upon analyses of proteolytically-derived or bacterially-expressed fragments have been refined by determination of the crystallographic structure of domain 1 in complex with actin [6], and that of fulllength gelsolin in submicromolar free Ca2+ [7••]. As expected, the conserved residues in each of the six domains define the basic domain structure, whereas actin binding and other functions involve residues that vary between the domains. The conformation of the protein is
Gelsolin family members and expression A new low abundance cytoplasmic gelsolin isoform, gelsolin-3, that differs from cytoplasmic gelsolin only by an additional 11 amino acid residues at its amino-terminus, has been identified [13]. Adseverin (scinderin) is the closest homologue to gelsolin and was originally discovered in bovine adrenal gland (Figure 2); however, in murine and human adult tissues, adseverin expression is highest in kidney and gut (>0.1% total cell protein in certain renal tubular epithelial cells), with lower levels in the thymus and adrenal gland (but not medulla), and very low to no expression elsewhere [14•]. Adseverin is also highly and variably expressed in hematopoietic lineage cells, where a novel isoform missing domain 5 (see Figure 2) was found [15•], and in endochondral bone primordia (Arai M, Kwiatkowski DJ, unpublished data). These observations conflict with the proposed models of a function for adseverin in exocytosis, at least in the majority of murine and human tissues [16]; moreover, the expression pattern of capG, adseverin and gelsolin is highly complementary in the mouse [14•] (Arai M, Kwiatkowski DJ, unpublished data), suggesting that the distinct functional characteristics of these proteins lead to distinct patterns of cellular expression according to the motile capacity of the cell. Human flightless, a gelsolin homologue with an amino-terminal leucine-rich repeat (LRR) (Figure 2) has been shown to bind to actin, and to bind to a newly described protein — flightless I LRR associated protein (FLAP) of size 626 amino acids — through its LRR [17•]. Advillin is a gelsolin family member that is most closely related to villin (59% amino-acid identity), including a headpiece domain [18•]. It is highly expressed in dorsal root and trigeminal ganglia during embryonic development and
cbb116.qxd
12/20/1999 12:23 PM
104
Page 104
Cytoskeleton
Figure 1
Ca2+/H+ Gelsolin Severing & capping
Nucleation & capping Uncapping
p
p p
A model of gelsolin’s interaction with actin in the cell. At rest, gelsolin is inactive, and depicted as a compact six domain structure. In response to micromolar Ca2+ or pH < 6.5, gelsolin opens up to become active in either severing or binding to actin monomers, resulting in a capped actin filament. In response to clustered phosphoinositides (zigzag-tailed hexagons) and possibly lysophosphatidic acid, gelsolin uncaps from the filament, and resumes a closed configuration in low Ca2+ and normal pH.
p
p
D3 and D4 phosphoinositides, ? lysophosphatidic acid Current Opinion in Cell Biology
at lower levels in adult uterine and intestinal epithelial cells. Advillin expression is induced in murine embryo fibroblasts in response to tunicamycin treatment and possibly other forms of cellular stress [19]. Supervillin (205 kDa) is another newly identified gelsolin family member that participates in interactions between actin filaments and membrane [20•]. It consists of an amino-terminal half which has nuclear targeting signals, and a carboxy-terminal half which in general has weak similarity to villin, but also contains more conserved regions corresponding to F-actin binding regions (Figure 2).
Gelsolin-related amyloidosis pathogenesis Recent evidence that gelsolin binds lysophosphatidic acid (LPA) [21••], which is present in diverse extracellular sites, suggests that an important function of secreted gelsolin is as a lipid carrier protein in addition to its postulated role in actin clearance [22]. Gelsolin-related amyloidosis is a rare hereditary amyloid polyneuropathy which is a result of Asp187→Asn or Asp187→Try mutations in gelsolin. These mutations cause an aberrant intracellular cleavage of secreted gelsolin between residues 172 and 173 leading to production of the amyloidogenic 173–243 fragment [23]. The 172/173 cleavage appears to be due to disruption of the Cys188–Cys201 disulfide bond in plasma gelsolin and to occur at highest frequency in neuronal cells, consistent with the pattern of organ involvement in this disease [24•].
The gelsolin-null mouse Gelsolin-null mice express no cytoplasmic or plasma gelsolin, and in mixed-strain backgrounds are completely viable with normal longevity under animal colony conditions [25]. They have proven to be a critical resource in the
analysis of gelsolin function in vivo [26••–28••,29•]. When the gelsolin gene defect is bred into either BALB/c or C57/black backgrounds, nearly all gelsolin-null pups die between embryonic day 17 and postnatal day 21 (Witke W, Kwiatkowski DJ, unpublished data), indicating that in concert with the multiple gene defects present in these strains, gelsolin is necessary for survival.
Gelsolin’s role in motility Analysis of dermal fibroblasts from the gelsolin-null mice has strengthened the view that gelsolin has a critical role in actin dynamics [26••]. The gelsolin-null cells display reduced ruffling activity and translocational motility in response to multiple stimuli and, in multiple assay systems, have increased amounts of F-actin in stress fibers and crawl by a distinctive pseudopod-like extension process. Both cultured cells and tissues from the gelsolinnull animals display a fivefold increase in the expression of the Rac GTPase, and the motility defect and Rac overexpression are repaired by gelsolin transfection. Filopodial protrusion is normal in these cells, suggesting that gelsolin serves a critical ‘effector’ function specific for cytokine motility signals transmitted by the Rac GTPase to the actin architecture (Figure 3). Interestingly, this effect is not seen in gelsolin-null embryo fibroblasts, consistent with different motility mechanisms during embryogenesis (Azuma T, Kwiatkowski DJ, unpublished data). Although gelsolin did not appear to be important in a cellfree system examining Listeria motility [30], gelsolin-transfected NIH3T3 cell lines displayed a proportional increase in Listeria motility [31•], consistent with a complex effect of gelsolin expression not amenable to cellfree analysis. The importance of gelsolin activity for motility has also been seen in gingival fibroblasts [32], and
cbb116.qxd
12/20/1999 12:23 PM
Page 105
Functions of gelsolin: motility, signaling, apoptosis, cancer Kwiatkowski
in epidermal growth factor receptor mediated motility in NR6 fibroblasts [33].
Figure 2 1
Signaling from Rac to gelsolin was also discovered in neutrophil extracts [34 •]. Release of actin from 1:1 gelsolin–actin complexes occurred independently of phosphatidylinositol 3-kinase and cellular phosphoinositides, and could be produced by GTP-loaded Rac or GTPloaded heterotrimeric G proteins. These observations contrast with models of gelsolin regulation derived from platelet studies [35], and may reflect the differing cell types and/or cell preparation methods. Movement of gelsolin to the Triton-X-100-insoluble cytoskeleton was observed during neutrophil adherence to fibronectin-coated plastic dishes [36].
2
3
4
5
6 gelsolin adseverin adseverin (D5) capG
LRR
flightless villin advillin
NT
Cultured embryonic hippocampal neurons were used to explore the function of gelsolin during neurite growth [29•]. Although ruffling activity and motility of growth cones were not affected, gelsolin-null neurites had an increased number of filopodia along their length. Filopodial extension was normal, but retraction was delayed, suggesting that gelsolin functioned to degrade the actin filament core during filopodial retraction.
Ion-channel regulation Modulation of ion-channel function by the actin cytoskeleton is increasingly understood [37,38], and the role of gelsolin in this process has now been shown in vivo [27••]. Gelsolin-null embryonic hippocampal neurons have increased filamentous actin, enhanced NMDA receptor-mediated currents, and enhanced Ca2+ intake and cell death in response to glutamate stimulation in comparison to wild-type neurons; moreover, seizureinduced damage to hippocampal neurons [27••] and ischemia-induced damage to the cortex (Endres M, Moscowitz MA, Kwiatkowski DJ, unpublished data) are both enhanced in gelsolin-null mice compared to wildtype animals.
Signaling and lipids Although originally viewed in the context of regulation of gelsolin function, the phosphoinositide–gelsolin interaction is now also recognized to modulate lipid signaling events. Gelsolin and capG binding to phosphoinositides is significantly enhanced by micromolar Ca2+ and also increased by mild acidification [39•]. The ability of gelsolin but not profilin to alter the structure of phosphatidylinositol 4,5-bisphosphate aggregates also reflects the high affinity of gelsolin for phosphoinositides [40•]; moreover, gelsolin and capG overexpression in NIH3T3 cells has significant effects on signaling through cytokine-stimulated pathways activating either phospholipase Cβ for phospholipase Cγ [41••]. Gelsolin has also been isolated in complexes with an increasing array of lipases and kinases, including phospholipases Cγ1, Cδ, and D, phosphatidylinositol 3-kinase, and
105
supervillin HP Current Opinion in Cell biology
Eight members of the gelsolin family of proteins (mammals only) are modeled. The core domain which is repeated six times is shown. LRR, leucine-rich repeat; HP, headpiece; NT, nuclear translocation domain.
c-src [42–44,45•,46•]. These results also suggest that gelsolin may alter lipid signaling pathways, either through direct binding to these proteins or, more likely (in my opinion), through joint binding to clustered phosphoinositides. Phosphatidylinositol 4,5-bisphosphate also enhances phosphorylation of gelsolin by the Src kinase to a maximum level of 0.18 mol phosphate per mol gelsolin in vitro [47], but there is no evidence for gelsolin phosphorylation in vivo. In osteoclasts, osteopontin stimulation induces formation of a signaling complex including gelsolin, phosphatidylinositol 3-kinase, and c-src, that is required for bone resorption [46•]. Gelsolin and other family members bind to LPA with high affinity [21••]. This interaction requires somewhat higher LPA concentrations than those needed for phosphatidylinositol 4,5-bisphosphate binding, but LPA also blocks gelsolin’s severing activity and uncaps gelsolin from filament ends. LPA acts cooperatively with phosphoinositides in inhibiting gelsolin’s interaction with actin and this provides another means for the regulation of gelsolin activity in cells.
Apoptosis Caspase-3 is a core effector protease in a protease cascade that is activated during apoptosis [48]. Gelsolin is a prominent substrate of caspase-3 in vitro [28••,49•], is cleaved during apoptosis in multiple cell types, and the amino-terminal cleavage product (residues 1–352) has Ca 2+ unregulated actin filament severing activity [28••,50]. Expression of this fragment in cells leads to apoptosis and gelsolin-null neutrophils progress to apoptotic cell death more slowly than wild-type neutrophils, consistent with an important role for the gelsolin
cbb116.qxd
12/20/1999 12:23 PM
106
Page 106
Cytoskeleton
Figure 3
Bradykinin
Cdc42
EGF PDGF Insulin
LPA
Rac
Rho Gelsolin
Filopodia
Ruffling Lamellipodia
Abundant actin stress fibers
family with distinctive expression patterns, domain structure and functions [13,15•,18•,20•]. Further insight into the mechanism of severing awaits the determination of gelsolin structure in activating conditions [7••]. Fibroblast and neutrophil studies indicate a signaling pathway from the Rac GTPase to gelsolin [26••,34•]. Gelsolin has unexpected roles in neurite filopodial retraction, neuronal ion channel modulation, and as a regulator and mediator of apoptosis [27 ••,28 ••, 29•,50,51••]. It also regulates phosphoinositide signaling [41••], and is regulated by and may modulate signaling by LPA [21••]. Gelsolin appears to be important in carcinogenesis [2,56••], but the mechanism is uncertain and could relate to its effects on motility, Rac or lipid signaling, and/or apoptosis.
Current Opinion in Cell Biology
Note added in proof A model for motility signaling within dermal fibroblasts. Cytokines signal for motility via three Rho family GTPases with differing effects. Gelsolin is an obligate effector of Rac-mediated motility and ruffling activity. In gelsolin-null cells, cytoplasmic sensing in some manner leads to upregulation of Rac expression which compensates somewhat but does not lead to normal motility.
The data referred to as Endres M, Moscowitz MA, Kwiatkowski DJ, unpublished data has now been published [57•].
Acknowledgements Supported by National Institutes of Health grants HL48743 and HL54188.
fragment in mediating apoptotic progression. Gelsolin, however, has also been found to inhibit apoptosis following several stimuli in Jurkat cells (a human T-cell line), when expressed at three to seven times normal levels [51••]. One possible resolution of these conflicting observations is that gelsolin, in complex with intracellular components such as phosphoinositides, might serve as a competitive inhibitor of caspase-3 (Azuma T, Kwiatkowski DJ, unpublished data).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Liu Y-T, Rozelle AL, Yin HL: The gelsolin family of actin filament severers and cappers. In G proteins, Cytoskeleton and Cancer. Edited by Maruta HK, Kohama K. Austin TX: RG Landes Company; 1998:19-35.
2.
Kuzumaki N, Fujita H, Tanaka M, Sakai N, Ohtsu M: Tumor suppressive function of gelsolin. In G Proteins, Cytoskeleton and Cancer. Edited by Maruta HK, Kohama K. Austin, TX: RG Landes Company; 1998:121-131.
3.
McGough A: F-actin-binding proteins. Curr Opin Struct Biol 1998, 8:166-176.
4.
Janmey PA, Stossel TP, Allen PG: Deconstructing gelsolin: identifying sites that mimic or alter binding to actin and phosphoinositides. Chem Biol 1998, 5:R81-R85.
5.
Kwiatkowski DJ, Stossel TP, Orkin SH, Mole JE, Colten HR, Yin HL: Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature 1986, 323:455-458.
6.
McLaughlin P, Gooch J, Mannherz H-G, Weeds A: Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature 1993, 364:685-692.
Cancer Gelsolin expression is downregulated in 60–90% of tumors during carcinogenesis [2] of the breast [52], colon, stomach, bladder [2], prostate [53], and lung [54•]. In addition, a Pro321→His mutation in gelsolin was identified as a suppressor of EJ-ras-mediated transformation of NIH3T3 cells: Pro321→His gelsolin and wild-type gelsolin inhibit the growth of ras-transformed NIH3T3 and a bladder cancer cell line, respectively, in the nude mouse [2]. Gelsolin expression is also increased by treatment of cells with either trichostatin A or radicicol, which have growth-suppressing, apoptosis-inducing activities [55•]. We have observed, however, that high level expression of gelsolin in early stage non-small-cell lung cancer occurs in 14% of patients, and provides highly significant negative prognostic information [56••]. In addition, the gelsolin-null mice show no increased incidence of tumors (Witke W, Kwiatkowski DJ, unpublished data).
7. ••
Burtnick LD, Koepf EK, Grimes J, Jones EY, Stuart DI, McLaughlin PJ, Robinson RC: The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation. Cell 1997, 90:661-670. The crystal structure of gelsolin in EGTA is presented. The modular domain structure of the protein is confirmed, and connector peptides identified. The structure is compact and a model for how it opens during Ca2+ activation is presented, although the structure in Ca2+ is not currently available. Residues that bind phosphoinositides are seen adjacent to those which bind to the side of an actin filament, explaining phosphoinositide-mediated uncapping and inhibition of severing. 8.
McGough A, Chiu W, Way M: Determination of the gelsolin binding site on F-actin: implications for severing and capping. Biophys J 1998, 74:764-772.
9.
Khaitlina S, Hinssen H: Conformational changes in actin induced by its interaction with gelsolin. Biophys J 1997, 73:929-937.
Conclusion Gelsolin-3, adseverin (D5 isoform), advillin, and supervillin are newly isolated members of the gelsolin
cbb116.qxd
12/20/1999 12:23 PM
Page 107
Functions of gelsolin: motility, signaling, apoptosis, cancer Kwiatkowski
10. Pope BJ, Gooch JT, Weeds AG: Probing the effects of calcium on gelsolin. Biochemistry 1997, 36:15848-15855. 11. Feinberg J, Kwiatek O, Astier C, Diennet S, Mery J, Heitz F, Benyamin Y, Roustan C: Capping and dynamic relation between domains 1 and 2 of gelsolin. J Pept Sci 1998, 4:116-127. 12. Hatanaka H, Ogura K, Moriyama K, Ichikawa S, Yahara I, Inagaki F: Tertiary structure of destrin and structural similarity between two actin-regulating protein families. Cell 1996, 85:1047-1055. 13. Vouyiouklis DA, Brophy PJ: A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system. J Neurochem 1997, 69:995-1005. 14. Lueck A, Brown D, Kwiatkowski DJ: The actin-binding proteins • adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine. J Cell Sci 1998, 111:3633-3643. In the mouse and humans, adseverin is found to be most highly expressed in certain renal tubular and intestinal epithelial cells, with lower levels in the thymus, adrenal gland (but not medulla), and uterus, and very low to no expression elsewhere. In addition, expression of adseverin and gelsolin in the kidney are complementary, occurring for the most part in different tubular epithelial cell populations. 15. Robbens J, Louahed J, De Pestel K, Van Colen I, Ampe C, • Vandekerckhove J, Renauld JC: Murine adseverin (D5), a novel member of the gelsolin family, and murine adseverin are induced by interleukin-9 in T-helper lymphocytes. Mol Cell Biol 1998, 18:4589-4596. In the mouse, adseverin was found to be widely expressed but at low levels, with higher amounts in hematopoietic cells, and significant induction during cytokine treatment in some cell populations. The D5 isoform, which is expressed only in hematopoietic cells, lacks the fifth and part of the sixth domain, but has normal Ca2+ activated severing activity, but no nucleating activity. 16. Zhang L, Marcu MG, Nau-Staudt K, Trifaro JM: Recombinant scinderin enhances exocytosis, an effect blocked by two scinderin-derived actin-binding peptides and PIP2. Neuron 1996, 17:287-296. 17. •
Liu YT, Yin HL: Identification of the binding partners for flightless I, a novel protein bridging the leucine-rich repeat and the gelsolin superfamilies. J Biol Chem 1998, 273:7920-7927. Human flightless I binds to actin–sepharose. The yeast two-hybrid method was used to identify flightless I LRR associated protein (FLAP), a binding partner of the flightless I LRR. FLAP is widely expressed and consists of 626 amino acids, but has unknown function. 18. Marks PW, Arai M, Bandura JL, Kwiatkowski DJ: Advillin (p92): a new • member of the gelsolin/villin family of actin regulatory proteins. J Cell Sci 1998, 111:2129-2136. A new member of the gelsolin family is described that is most closely homologous to villin. Advillin shares the six domain structure of other gelsolin family members and, in addition, has a carboxy-terminal headpiece domain that is similar to the headpiece domain of villin. Advillin is expressed at highest levels in developing and adult ganglia in the mouse, with much lower levels expressed in the uterus and intestine, and little to none elsewhere. 19. Wang XZK, Kuroda M, Sok J, Batchvarova N, Kimmel R, Chung P, Zinszner H, Ron D: Identification of novel stress-induced genes downstream of chop. EMBO J 1998, 17:3619-3630. 20. Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ: Supervillin • (p205): a novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily. J Cell Biol 1997, 139:1255-1269. Supervillin is purified and cloned. It is a 205 kDa protein that purifies with neutrophil plasma membranes and binds to the sides of actin filaments in overlay assays. The protein is found in different intracellular locations in neutrophils, HeLa and MDCK cells, depending upon culture conditions and treatments. Structurally it consists of an amino-terminal half with apparent nuclear localization function, and a carboxy-terminal half with weak overall similarity to villin, and stronger similarity in regions of actin-binding activity.
107
23. Maury CP, Sletten K, Totty N, Kangas H, Liljestrom M: Identification of the circulating amyloid precursor and other gelsolin metabolites in patients with G654A mutation in the gelsolin gene (Finnish familial amyloidosis): pathogenetic and diagnostic implications. Lab Invest 1997, 77:299-304. 24. Paunio T, Kangas H, Heinonen O, Buc-Caron MH, Robert JJ, • Kaasinen S, Julkunen I, Mallet J, Peltonen L: Cells of the neuronal lineage play a major role in the generation of amyloid precursor fragments in gelsolin-related amyloidosis. J Biol Chem 1998, 273:16319-16324. The authors disrupt the Cys188–Cys201 disulfide bond in gelsolin by sitedirected mutagenesis and find that aberrant intracellular processing occurs, similar to that seen in Asp187→Asn amyloidogenic gelsolin. The implication is that the Asp187→Asn mutation disrupts this disulfide bond, leading to cleavage. In addition, this intracellular cleavage occurs at a higher rate in neuronal lineage cells than in other cell types. 25. Witke W, Sharpe AH, Hartwig JH, Azuma T, Stossel TP, Kwiatkowski DJ: Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell 1995, 81:41-51. 26. Azuma T, Witke W, Stossel TP, Hartwig JH, Kwiatkowski DJ: Gelsolin •• is a downstream effector of Rac for fibroblast motility. EMBO J 1998, 17:1362-1370. Gelsolin-null dermal fibroblasts are characterized in detail in comparison to wild-type fibroblasts. The gelsolin-null cells have increased F-actin, and reduced motility and pinocytosis in several assay systems. They crawl using a distinctive filopodia-like structure in contrast to the ruffles and lamellipodia present in the wild-type cells. Both the gelsolin-null cells and tissues from the gelsolin-null mice show expression of Rac at fivefold higher levels than wild-type cells, and the motility defect and Rac overexpression are reverted to wild-type by transfection of gelsolin. The implication is that gelsolin is an obligate element downstream of the Rac GTPase for motility. 27. ••
Furukawa K, Fu W, Li Y, Witke W, Kwiatkowski DJ, Mattson MP: The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J Neurosci 1997, 17:8178-8186. Primary hippocampal gelsolin-null neurons have decreased actin filament depolymerization and increased Ca2+ influx in response to glutamate compared to wild-type neurons. Enhanced currents in response to NMDA were also seen in patch-clamp experiments, as a result of a lack of current rundown. Cultured neurons showed greater cell death in response to high-dose glutamate, and seizure-induced damage to hippocampal pyramidal neurons was increased in adult gelsolin-null mice. The implication is that gelsolin has an important role in modulating neuronal ion channel function in response to excitotoxicity in vivo. 28. Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, •• McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ, Williams LT: Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 1997, 278:294-298. Screening of 100,000 murine embryo fibroblast cDNA clones in an in vitro transcription-translation reaction led to identification of gelsolin as the most prominent substrate of caspase-3. The amino-terminal gelsolin fragment generated by caspase-3 cleavage has unregulated actin filament severing activity, and when expressed in cells causes apoptosis. Gelsolin-null neutrophils have a slower rate of apoptotic death in response to tumor necrosis factor induction, compared to wild-type neutrophils. The implication is that gelsolin has an important role in apoptotic progression. 29. Lu M, Witke W, Kwiatkowski DJ, Kosik KS: Delayed retraction of • filopodia in gelsolin null mice. J Cell Biol 1997, 138:1279-1287. Although growth cone extension, ruffling activity, and translocational rates are identical in cultured gelsolin null neurons compared to wild-type, the null neurons have increased numbers of neurite filopodia. This is due to delayed filopodial retraction that implicates a function of gelsolin in dissassembling the F-actin rich core of the filopodia. 30. Rosenblatt J, Agnew BJ, Abe H, Bamburg JR, Mitchison TJ: Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails [see comments]. J Cell Biol 1997, 136:1323-1332.
21. Meerschaert K, De Corte V, De Ville Y, Vandekerckhove J, •• Gettemans J: Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid. EMBO J 1998, 17:5923-5932. An extensive survey of phospholipids led to the discovery that gelsolin binds to lysophosphatidic acid (LPA). LPA binding affects gelsolin function in a manner similar to phosphoinositides, but requires significantly higher concentrations of LPA. LPA has synergistic effects on gelsolin function when added to phosphoinositides; therefore, LPA may act as another intracellular component that regulates gelsolin activity on the actin cytoskeleton. Secreted gelsolin may also serve as a carrier of LPA and perhaps other phospholipids, significantly affecting their presentation to and function on cells.
31. Laine RO, Phaneuf KL, Cunningham CC, Kwiatkowski D, Azuma T, • Southwick FS: Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells. Infect Immun 1998, 66:3775-3782. Listeria motility and actin tail lengths were both significantly increased in NIH3T3 cells that had been transfected to overexpress gelsolin, and the increases were proportional to the level of gelsolin expression.
22. Suhler E, Lin W, Yin HL, Lee WM: Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock, and myonecrosis. Crit Care Med 1997, 25:594-598.
32. Arora PD, McCulloch CAG: Dependence of fibroblast migration on actin severing activity of gelsolin. J Biol Chem 1996, 271:20516-20523.
cbb116.qxd
12/20/1999 12:23 PM
108
Page 108
Cytoskeleton
33. Chen P, Murphy-Ullrich JE, Wells A: A role for gelsolin in actuating epidermal growth factor receptor-mediated cell motility. J Cell Biol 1996, 134:689-698.
oligonucleotides against c-Src led to reduced c-Src expression, reduced association between gelsolin and c-Src in osteoclast extracts, reduced associated phosphatidylinositol 3-kinase activity and reduced bone resorption.
34. Arcaro A: The small GTP-binding protein Rac promotes the • dissociation of gelsolin from actin filaments in neutrophils. J Biol Chem 1998, 273:805-813. Gelsolin–actin complexes are present in resting neutrophils and are dissociated by fMLP (f-Met-Leu-Phe, a chemotactic peptide for neutrophils) activation. Here this effect is shown to be insensitive to wortmannin or kinase inhibitor treatment. In a cell-free system, gelsolin–actin dissociation was achieved by addition of GTP-loaded Rac or heterotrimeric G proteins, implicating a signaling pathway between Rac and gelsolin that is independent of phosphoinositides.
47.
35. Barkalow K, Witke W, Kwiatkowski DJ, Hartwig JH: Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein. J Cell Biol 1996, 134:389-399. 36. Wang JS, Coburn JP, Tauber AI, Zaner KS: Role of gelsolin in actin depolymerization of adherent human neutrophils. Mol Biol Cell 1997, 8:121-128. 37.
Cantiello H: Role of actin filament organization in cell volume and ion channel regulation. J Exp Zool 1997, 279:425-435.
38. Lascola CD, Nelson DJ, Kraig RP: Cytoskeletal actin gates a Clchannel in neocortical astrocytes. J Neurosci 1998, 18:1679-1692. 39. Lin KM, Wenegieme E, Lu PJ, Chen CS, Yin HL: Gelsolin binding to • phosphatidylinositol 4,5-bisphosphate is modulated by calcium and pH. J Biol Chem 1997, 272:20443-20450. Superdex gel filtration was used as a primary assay to demonstrate that micromolar Ca2+ increases the affinity of gelsolin (eightfold) and capG (fourfold) for phosphatidylinositol 4,5-bisphosphate. Lowering the pH from 7.5 to 7.0 reduced the level of Ca2+ required for this effect. The implication is that during cell activation, the affinity of gelsolin and capG for phosphoinositides is increased, potentially altering phosphoinositides signaling pathways. 40. Flanagan LA, Cunningham CC, Chen J, Prestwich GD, Kosik KS, • Janmey PA: The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and tau. Biophys J 1997, 73:1440-1447. Divalent cations cause phosphatidylinositol 4,5-bisphosphate to aggregate into clusters of striated filaments. Gelsolin and tau, but not profilin, affected the structure of these aggregates, confirming gelsolin’s high affinity binding and suggesting it can alter phosphatidylinositol 4,5-bisphosphate distribution in the cell membrane. 41. Sun H, Lin K, Yin HL: Gelsolin modulates phospholipase C activity •• in vivo through phospholipid binding. J Cell Biol 1997, 138:811-820. Utilizing NIH3T3 cells stably transfected to overexpress gelsolin or capG, it is shown that overexpression of either protein strongly inhibits bradykininstimulated phospholipase Cβ and weakly inhibits tyrosine kinase-stimulated phospholipase Cγ. Specificity of the gelsolin effect on phospholipase Cβ was confirmed in washout and addback experiments performed on permeabilized cells. The implication is that these proteins can significantly affect phosphoinositide-signaling pathways in cells. 42. Banno Y, Nakashima T, Kumada T, Ebisawa K, Nonomura Y, Nozawa Y: Effects of gelsolin on human platelet cytosolic phosphoinositidephospholipase C isozymes. J Biol Chem 1992, 267:6488-6494. 43. Steed PM, Nagar S, Wennogle LP: Phospholipase D regulation by a physical interaction with the actin- binding protein gelsolin. Biochemistry 1996, 35:5229-5237. 44. Singh SS, Chauhan A, Murakami N, Chauhan VP: Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity. Biochemistry 1996, 35:16544-16549. 45. Baldassare JJ, Henderson PA, Tarver A, Fisher GJ: Thrombin • activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cγγ1. Biochem J 1997, 324:283-287. Co-immunoprecipitation experiments demonstrate that phospholipase Cγ is associated with a gelsolin–actin complex in fresh unstimulated platelets, and that this three-protein complex dissolves in response to thrombin stimulation, requiring platelet aggregation and tyrosine phosphorylation events. Phospholipase Cγ activity increases concurrent with release from gelsolin. 46. Chellaiah M, Fitzgerald C, Alvarez U, Hruska K: c-Src is required for • stimulation of gelsolin-associated phosphatidylinositol 3-kinase. J Biol Chem 1998, 273:11908-11916. Osteopontin stimulation of osteoclasts induces binding of c-Src to gelsolin in co-immunoprecipitation assays, in addition to phosphatidylinositol 3-kinase (as shown previously by these authors). Treatment of osteoclasts with antisense
De Corte V, Gettemans J, Vandekerckhove J: Phosphatidylinositol 4,5-bisphosphate specifically stimulates PP60(c- src) catalyzed phosphorylation of gelsolin and related actin-binding proteins. FEBS Lett 1997, 401:191-196.
48. Cryns V, Yuan J: Proteases to die for. Genes Dev 1998, 12:1551-1570. 49. Kamada S, Kusano H, Fujita H, Ohtsu M, Koya RC, Kuzumaki N, • Tsujimoto Y: A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA 1998, 95:8532-8537. A mutant caspase-3 was used in the yeast two-hybrid system to identify binding partners of caspase-3. Gelsolin was identified as a prominent substrate, and its cleavage confirmed in vitro. 50. Geng Y-JA T, Tang JX, Hartwig JH, Muszynski M, Libby P, Kwiatkowski DJ: Caspase-3 induced gelsolin fragmentation contributes to actin cytoskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle cells exposed to proinflammatory cytokines. Eur J Cell Biol 1999, 50:294-302. 51. Ohtsu M, Sakai N, Fujita H, Kashiwagi M, Gasa S, Shimizu S, •• Eguchi Y, Tsujimoto Y, Sakiyama Y, Kobayashi K, Kuzumaki N: Inhibition of apoptosis by the actin-regulatory protein gelsolin. EMBO J 1997, 16:4650-4656. Stable gelsolin-overexpressing transfectant lines were derived from the Jurkat cell line, a human T-cell line. These cell lines were found to have marked resistance to apoptotic induction to anti-fas antibodies, C2-ceramide, and dexamethasone, and a marked reduction in the production of caspase-3 activity. The implication is that gelsolin is blocking apoptosis at some critical and universal early step in these cells. 52. Asch HL, Head K, Dong Y, Natoli F, Winston JS, Connolly JL, Asch BB: Widespread loss of gelsolin in breast cancers of humans, mice, and rats. Cancer Res 1996, 56:4841-4845. 53. Prasad SC, Thraves PJ, Dritschilo A, Kuettel MR: Protein expression changes associated with radiation-induced neoplastic progression of human prostate epithelial cells. Electrophoresis 1997, 18:629-637. 54. Dosaka-Akita H, Hommura F, Fujita H, Kinoshita I, Nishi M, • Morikawa T, Katoh H, Kawakami Y, Kuzumaki N: Frequent loss of gelsolin expression in non-small cell lung cancers of heavy smokers. Cancer Res 1998, 58:322-327. Gelsolin expression was reduced in twelve out of twelve human lung cancer cell lines — markedly so (<10% normal levels) in ten of the twelve. Of 88 primary resected tumors 48 (55%) had no gelsolin expression, and there was no correlation with histologic type or stage. 55. Kwon HJ, Yoshida M, Nagaoka R, Obinata T, Beppu T, Horinouchi S: • Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression. Oncogene 1997, 15:2625-2631. Radicicol, an inhibitor of src-family kinases, reverts the morphology of transformed fibroblasts to untransformed fibroblasts. In two-dimensional gel analyses, gelsolin was identified as being prominently upregulated in radicicol-treated cells, similar to what the authors have seen previously with trichostatin A treatment. Injection of anti-gelsolin antibodies inhibited the morphologic reversion induced by radicicol in T24 and HeLa cells. 56. Shieh DGJ, Sugarbaker DJ, Herndon J, Kwiatkowski DJ: Motility as a •• prognostic factor in non-small cell lung cancer: role of gelsolin expression. Cancer 1999, 85:47-57. Analysis of histologic sections from 229 patients with stage I non-small cell lung cancer was performed. Rac and ABP-280 expression (an actin filament crosslinking protein, also known as filamin) appeared to have no influence on prognosis in a pilot study, but gelsolin expression was important. The 32 patients who expressed gelsolin highly (either uniformly or focally in their tumors) had a fourfold higher relative risk of cancer recurrence, and gelsolin expression was a greater risk factor than any other variable examined. 57. •
Endres M, Fink K, Zhu J, Stagliano NE, Bondala V, Geddes JW, Azuma T, Mattson MP, Kwiatkowski DJ, Moscowitz MA: Neuroprotective effects of gelsolin and cytochalasin D during murine stroke. J Clin Invest 1999, in press. Cultured gelsolin-null neurons have enhanced cell death and sustained elevation of Ca2+ levels after glucose/oxygen deprivation. In addition, infarct volumes are 36% greater in gelsolin-null mice than wild-type controls after transient middle cerebral artery occlusion. This increased stroke volume is eliminated by intraventricular injection of cytochalasin D, which also decreases stroke volume in wild-type mice. The implication is that modulation of the actin filament architecture by gelsolin and analogues such as cytochalasin D may improve the outcome from stroke.