Biology subjects

Turkewitz, A.

Publications and source records attributed to Turkewitz, A..

4 recordsLinked to original sources

bioRxiv2026

Tet Trim-Away: A conditional rapid protein degradation system for Tetrahymena thermophila

Tetrahymena thermophila is a ciliated protist that has played pivotal roles in biological discovery. Functional studies of Tetrahymena proteins have largely relied on gene knockouts. Because protein depletion upon knockout typically spans multiple cell cycles, compensatory mechanisms can confound phenotypic interpretation. To instead enable rapid and acute protein depletion, we modified and adapted the Trim-Away system for use in Tetrahymena (Tet Trim-Away). Trim-Away is based on the E3 ubiquitin ligase, TRIM21, that binds to antibody-bound proteins and targets them for proteasome mediated degradation. Here, Trim-Away was modified with a fusion of the N-terminal RBCC (RING, B-box, coiled-coil) domains of TRIM21 with an -mCherry (mCh) nanobody sequence that recognizes endogenously tagged mCh proteins of interest (NbmCh). Expression of the RBCC:NbmCh degron, which is controlled by an inducible promotor, promotes rapid target protein depletion within 30 minutes and can be sustained for weeks. Tet Trim-Away is reversible, functions against targets in multiple cellular compartments, and produces loss-of-function phenotypes in Tetrahymena cells.

cell biology↗

bioRxiv2023

VPS8D, a CORVET subunit, is required to maintain the contractile vacuole complex in Tetrahymena thermophila

Contractile vacuole complexes (CVCs) are complex osmoregulatory organelles, with vesicular (bladder) and tubular (spongiome) subcompartments. The mechanisms that underlie their formation and maintenance within the eukaryotic endomembrane network are poorly understood. In the Ciliate Tetrahymena thermophila, six differentiated CORVETs (class C core vacuole/endosome tethering complexes), with Vps8 subunits designated A-F, are likely to direct endosomal trafficking. Vps8Dp localizes to both bladder and spongiome. We show by inducible knockdown that VPS8D is essential to CVC organization and function. VPS8D knockdown increased susceptibility to osmotic shock, tolerated in the wildtype but triggering irreversible lethal swelling in the mutant. The knockdown rapidly triggered contraction of the spongiome and lengthened the period of the bladder contractile cycle. More prolonged knockdown resulted in disassembly of both the spongiome and bladder, and dispersal of proteins associated with those compartments. In stressed cells where the normally singular bladder is replaced by numerous vesicles bearing bladder markers, Vps8Dp concentrated conspicuously at long-lived inter-vesicle contact sites, consistent with tethering activity. Similarly, Vps8Dp in cell-free preparations accumulated at junctions formed after vacuoles came into close contact. Also consistent with roles for Vps8Dp in tethering and/or fusion were the emergence in knockdown cells of multiple vacuole-related structures, replacing the single bladder. SynopsisIn the Ciliate Tetrahymena thermophila, VPS8D, which encodes a subunit of a non-conventional CORVET complex, is an essential determinant of the contractile vacuole complex (CVC). VPS8D knockdown results in retraction and dispersal of the spongiome, and disappearance of the bladder, reinforcing the view that CVCs arise from endosomal trafficking. Intermediate knockdown phenotypes and Vps8Dp localization support a role in homotypic tethering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/566071v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b652c6org.highwire.dtl.DTLVardef@1f456f7org.highwire.dtl.DTLVardef@79ab8dorg.highwire.dtl.DTLVardef@1edbf74_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Structure and dynamics of the contractile vacuole complex in Tetrahymena thermophila

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprised of a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying the structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, while the lipid scramblase Scr7p co-localizes with Vps8Dp. The H+- ATPase subunit Vma4 is spongiome-specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools vs. lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. While the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome are isolated from bulk endocytic input. Summary statementIn the ciliate Tetrahymena thermophila, four proteins are shown to provide markers for different zones of the contractile vacuole complex. They shed light on its formation and maintenance by enabling in vivo analysis of its dynamics.

cell biology↗

bioRxiv2019

Diversification of CORVET tethers facilitates transport complexity in Tetrahymena thermophila

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking genes while retaining the related CORVET complex. Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal vs. recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how backfilling following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways.

cell biology↗