Stable and dynamic axes of polarity use distinct formin isoforms in budding yeast - PubMed (original) (raw)

Stable and dynamic axes of polarity use distinct formin isoforms in budding yeast

David Pruyne et al. Mol Biol Cell. 2004 Nov.

Abstract

Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell.

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Figures

Figure 1.

Figure 1.

Bni1p and Bnr1p are important for distinct sets of actin cables. (A) Wild-type cells (Y1239) were grown 2 h in galactose to induce a control vector or NH2-terminally truncated Bni1p (BNI1_Δ_N) or Bnr1p (BNR1_Δ_N) and stained for Act1p distribution by immunofluorescence microscopy. (B) Recombinant GST (190 nM) or GSTBnr1p(FH1FH2) (60 or 120 nM) were combined with 3 μM G-actin (∼5% pyrene labeled), and actin assembly was monitored through increases in pyrene fluorescence (measured in arbitrary units). (C) Models depict the direction of polarized transport in yeast at various stages of bud growth that correspond with cell images below. Bni1p is shown in selected wild-type cells (ABY1848) treated for anti-Bni1p immunofluorescence or expressing Bni1pGFP from a high-copy plasmid. Bnr1pGFP fluorescence is shown in selected wild-type cells (YEF2255). The distribution of actin cables in selected wild-type (ABY1848), bnr1_Δ/bnr1_Δ (ABY1801), and bni1_Δ/bni1_Δ (ABY1867) cells is shown by anti-Tpm1p immunofluorescence microscopy. (D) One hundred cells of each indicated budding categories of wild-type (ABY1848), bnr1_Δ/bnr1_Δ (ABY1801), and bni1_Δ/bni1_Δ (ABY1867) strains were processed for localization of Tpm1p by immunofluorescence microscopy as a reporter for actin cable organization. Unbudded cells scored as either having organized cables (black) or disorganized cables (blank). Budded cells were categorized as having unorganized cables (blank), cables predominantly associated with the bud cortex (black), cables associated with bud cortex and neck (gray), or cables predominantly associated with the bud neck (white).

Figure 2.

Figure 2.

Myo2p localization is perturbed in _bni1_Δ cells. (A) Models as in Figure 1A are depicted with selected wild-type (ABY1848), bnr1_Δ/bnr1_Δ (ABY1801), and bni1_Δ/bni1_Δ (ABY1867) cells stained to show Myo2p distribution. (B) One hundred cells of each budding category treated as described in A were scored for Myo2p localization. Unbudded cells were categorized as having Myo2p delocalized (blank) or localized to one site on the cell surface (black). Budded cells were scored as having Myo2p localized to the bud cortex (though not necessarily the bud tip) (black), the bud cortex and neck (gray), localized to the bud neck (white), or delocalized (blank).

Figure 3.

Figure 3.

_Bni1_Δ and _bnr1_Δ yeast have unique perturbations in vesicle dynamics. (A) Still GFPSec4p fluorescence images from movies of living wild-type (ABY1848; Mov. S1), bni1_Δ/bni1_Δ (ABY1867; Mov. S2), and bnr1_Δ/bnr1_Δ (ABY1801; Mov. S3) cells are shown. (B) Results of density counts for individual fluorescent particles from individual frames from 19 movies of GFPSec4p-expressing small- or medium-budded cells of each strain in A are summarized. The density of discrete particles counted in the indicated portions of each mother cell was plotted against the number of times that density was counted (with 315 total counting events for mother cell-half for each strain).

Figure 4.

Figure 4.

Actin cable filaments assemble at the bud cortex and neck. (A) Tpm1-2/tpm1-2 tpm2_Δ/tpm2_Δ mutants (ABY971) were subjected to the indicated conditions and stained to show Tpm1p and Myo2p distributions. (B) Tpm1-2/tpm1-2 tpm2_Δ/tpm2_Δ cells (ABY971) were shifted to 34.5°C for varying lengths of time before restoration to 18°C for 1 min and treatment for anti-Myo2p immunofluorescence microscopy. One hundred medium-budded cells from each time point were scored for the presence of Myo2p at the bud tip (gray squares) or neck (black circles).

Figure 5.

Figure 5.

Bni1p and Bnr1p differentially direct bud cortex- and bud neck-associated filament assembly. (A) Strains bearing _tpm1-2 tpm2_Δ (ABY944), _tpm1-2 tpm2_Δ _bnr1_Δ (ABY1804), _tpm1-2 tpm2_Δ bni1-12 (ABY1805), and _tpm1-2 tpm2_Δ _bni1-12 bnr1_Δ (ABY1806) mutations were subjected to the indicated conditions and stained to show Tpm1p distribution. (B) One hundred medium-budded cells of each strain in A were shifted to 34.5°C for the indicated times before restoration to 18°C for 1 min and staining to show Tpm1p distribution. Cells were scored for the presence of Tpm1p in the bud (black), in the bud and at the neck (gray), at the neck (white), or delocalized (blank). Results for small-budded cells were similar.

Figure 6.

Figure 6.

Septins localize Bnr1p-dependent cable assembly to the bud neck. (A) Diploid wild-type (ABY1848), bni1-11/bni1-11 (ABY2247), cdc10-1/cdc10-1 (ABY1662), bni1-11/bni1-11 cdc10-1/cdc10-1 (ABY2248), bni1-11/bni1-11 bnr1_Δ/bnr1_Δ (ABY1803), and tpm1-2/tpm1-2 tpm2_Δ/tpm2_Δ (ABY2250) were shifted to 35°C for 1 h and stained to show Tpm1p distribution. Corresponding haploid strains (Y1239, ABY2209, ABY2201, ABY2226, ABY2218, and ABY1807) were shifted to 35°C for 4 h and observed by differential interference contrast microscopy. (B) _Tpm1-2 tpm2_Δ (ABY944), _tpm1-2 tpm2_Δ cdc10-1 (ABY1639), and _tpm1-2 tpm2_Δ cdc12-6 (ABY1643) cells were subjected to indicated conditions and stained for Tpm1p distribution. (C) Cells of each strain in B were shifted to 34.5°C for the indicated times before 1 min restoration to 18°C and staining to show Tpm1p distribution. One hundred medium-budded cells were scored for the presence of Tpm1p in the bud (black), in the bud and at the neck (gray), at the neck (white), or delocalized (blank). Results for small-budded cells were similar. (D) Bnr1pGFP-fluorescence is shown for wild-type (YEF2255) and cdc10-1/cdc10-1 mutant cells (ABY2262) after 1 h at 34.5°C. (E) Fifty cells each of unbudded (unbud.) and small- (small), medium- (med.), and large-budded (large) cells treated as in D were assayed for the localization of Bnr1pGFP to a single point on the cell cortex (for unbudded cells) or the bud neck (for budded cells).

Figure 7.

Figure 7.

Inhibition of the switch from apical to isotropic bud growth does not preserve bud cortex-associated cable assembly in the absence of actin cables. (A) _GAL-CLN2_–bearing wild-type (ABY2251) and _tpm1-2 tpm2_Δ (ABY 2252) cells either were (+Gal) or were not (-Gal) induced to overproduce Cln2p for 4 h at the indicated temperatures and observed by differential interference contrast microscopy or stained to show Tpm1p distribution. Arrows indicate bud tips with associated Tpm1p. (B) Wild-type (Y1239) or _GAL-CLN2_-bearing (ABY2251) cells induced for 4 h were grown at 18°C and stained for Myo2p. _Tpm2_Δ (ABY945), _cdc10-1 tpm2_Δ (ABY1637), and _cdc12-6 tpm2_Δ (AY1639) cells were grown at 35°C for 1 h before staining to show Myo2p distribution. One hundred cells of each strain with medium-sized and/or elongated buds were scored for the presence of Myo2p at the bud tip (black), at the bud tip and the neck (gray), at the bud neck (white), or not localized (blank). (C) _Tpm1-2 tpm2_Δ GAL-CLN2 cells (ABY2252) were subjected to the indicated conditions and stained to show Myo2p distribution. (D) _Tpm1-2 tpm2_Δ (ABY944) cells, _tpm1-2 tpm2_Δ GAL-CLN2 (ABY2252) cells grown 4 h in galactose, _tpm1-2 tpm2_Δ cdc10-1 (ABY1639) cells, and _tpm1-2 tpm2_Δ cdc12-6 (ABY1643) cells were shifted to 34.5°C for 2 or 60 min before restoration to 18°C for 1 min and Myo2p localized by immunofluorescence microscopy. One hundred medium-budded cells of each were scored for the presence of Myo2p at the bud tip (black), at the bud tip and neck (gray), at the bud neck (white), or not localized (blank).

Figure 8.

Figure 8.

Disruption of polarized secretion depletes bud-associated cables. (A) Wild-type (ABY501), myo2-16/myo2-16 (ABY506), sec2-41/sec2-41 (ABY1665), and sec16-2 (RSY267) cells were subjected to the indicated conditions and stained to show Tpm1p distribution. (B) Wild-type (ABY501), myo2-16/myo2-16 (ABY506), sec2-41/sec2-41 (ABY1665), sec4-8/sec4-8 (ABY994), sec6-4/sec6-4 (ABY993), sec10-2/sec10-2 (ABY995), sec15-1/sec15-1 (ABY996), sec16-2 (RSY267), and sec23-1 (RSY281) cells were subjected to the indicated conditions and stained to show Myo2p distribution. (C) One hundred medium-budded cells of each sample in B plus the wild-type haploid RSY255 were scored for the presence of Myo2p at the bud tip (black), the bud tip and neck (gray), the neck (white), or delocalized (blank). Results were similar for small-budded cells.

Figure 9.

Figure 9.

Rho-proteins, but not other cable-assembly factors, strongly depend on cables for retention at the bud tip. (A) Tpm1-2/tpm1-2 tpm2_Δ/tpm2_Δ strains expressing Spa2pGFP (ABY1197), GFPBud6p (ABY1171), HARho1p (ABY1602), Bnr1pGFP (ABY1891), GFPCdc12p (ABY1899), or no epitope-tagged protein (ABY971) were subjected to the indicated growth conditions and stained to show Bni1p, GFP, Cdc42p, and HA-epitope distributions. Examples of aberrant Bni1p, Spa2pGFP, GFPBud6p, Cdc42p, and HARho1p localization at 34.5°C are shown. (B) Small- and medium-budded cells of each sample in A plus identically treated TPM1/TPM1 tpm2_Δ/tpm2_Δ control strains expressing Spa2pGFP (ABY1198), GFPBud6p (ABY1199), HARho1p (ABY1601), GFPCdc12p (ABY1896), or no epitope-tagged protein (ABY973), or TPM1/TPM1 TPM2/TPM2 Bnr1pGFP control strain (YEF2255), were scored for the presence of stain at the bud tip (black), as puncta along the bud cortex (speckled), at the bud tip and neck (gray), at the neck (white), or delocalized (blank). For strains ABY1171, 1197, 1198, 1199, 1601, and 1602, 200 small- and 200 medium-budded cells of each were scored and averaged. For the remaining strains, 100 small- and 100 medium-budded cells of each strain were scored and averaged.

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