Cynthia Y. He

Golgi biogenesis in Toxoplasma gondii

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.

Golgi duplication in Trypanosoma brucei

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.

Golgi Duplication in Trypanosoma brucei Requires Centrin2

Centrins are highly conserved components of the centrosome, which in the parasitic protozoan T. brucei comprises the basal body and nucleates the flagellum used for locomotion. Here, we found TbCentrin2 in an additional bi-lobed structure near to the Golgi apparatus. One lobe was associated with the old Golgi, and the other became associated with the newly forming Golgi as the cell grew. Depletion of TbCentrin1 inhibited duplication of the basal body, whereas depletion of TbCentrin2 also inhibited duplication of the Golgi. Thus, a Centrin2-containing structure distinct from the basal body appears to mark the site for new Golgi assembly.

Local inflammation induces complement crosstalk which amplifies the antimicrobial response.

By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection.

Ordered assembly of the duplicating Golgi in Trypanosoma brucei

The new Golgi in the protozoan parasite Trypanosoma brucei grows near to the old and adjacent to the growing new endoplasmic reticulum exit site. Growth is now shown to be at least a two-stage process, in which a representative matrix marker (GRASP) and enzyme (GntB) are delivered to the site of assembly, followed ≈10 min later by a COPI component (ε-COP) and a trans-Golgi network (TGN) marker (GRIP70). A secretory cargo marker (signal sequence-YFP) appeared early near the new endoplasmic reticulum exit site but did not enter the Golgi until the second stage. Together these data suggest that structural and enzymatic components of the new Golgi stack are laid down first, followed by those needed to move and sort the cargo passing through it.

A coiled-coil- and C2-domain-containing protein is required for FAZ assembly and cell morphology in Trypanosoma brucei

Trypanosoma brucei, a flagellated protozoan parasite causing human sleeping sickness, relies on a subpellicular microtubule array for maintenance of cell morphology. The flagellum is attached to the cell body through a poorly understood flagellum attachment zone (FAZ), and regulates cell morphogenesis using an unknown mechanism. Here we identified a new FAZ component, CC2D, which contains coiled-coil motifs followed by a C-terminal C2 domain. T. brucei CC2D is present on the FAZ filament, FAZ-juxtaposed ER membrane and the basal bodies. Depletion of CC2D inhibits the assembly of a new FAZ filament, forming a FAZ stub with a relatively fixed size at the base of a detached, but otherwise normal, flagellum. Inhibition of new FAZ formation perturbs subpellicular microtubule organization and generates short daughter cells. The cell length shows a strong linear correlation with FAZ length, in both control cells and in cells with inhibited FAZ assembly. Together, our data support a direct function of FAZ assembly in determining new daughter cell length by regulating subpellicular microtubule synthesis.

A Comparative Proteomic Analysis Reveals a New Bi-Lobe Protein Required for Bi-Lobe Duplication and Cell Division in Trypanosoma brucei

A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe) and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component. The anterior part of the TbLRRP1-labeled bi-lobe is adjacent to the single Golgi apparatus, and the posterior side is tightly associated with the flagellar pocket collar marked by TbBILBO1. Inducible depletion of TbLRRP1 by RNA interference inhibited duplication of the bi-lobe as well as the adjacent Golgi apparatus and flagellar pocket collar. Formation of a new flagellum attachment zone and subsequent cell division were also inhibited, suggesting a central role of bi-lobe in Golgi, flagellar pocket collar and flagellum attachment zone biogenesis.

Centrin4 coordinates cell and nuclear division in T. brucei

Centrins are Ca2+-binding proteins that have been implicated in a number of biological processes, including organelle duplication, mRNA export, DNA repair and signal transduction. In the protozoan parasite Trypanosoma brucei we have previously described TbCentrin2, which is present on a bi-lobed structure, and involved in the duplication and segregation of the Golgi complex. Recently, another centrin, TbCentrin4, was also found at the bi-lobe and has been implicated in organelle segregation and cytokinesis. We now show that cytokinesis is not inhibited, but that a dysregulation of nuclear and cell division leads to the production of zoids – daughter siblings that contain all organelles except the nucleus. Our results, therefore, suggest that TbCentrin4 is involved in processes that coordinate karyokinesis and cytokinesis.

Golgi biogenesis in simple eukaryotes

The accurate duplication of cellular organelles is important to ensure propagation through successive generations. The semi‐conserved replication of DNA and DNA‐containing organelles has been well studied, but the mechanisms used to duplicate most other organelles remain elusive. These include the centrosomes, which act as microtubule organizing centres during interphase and orient the mitotic spindle poles during mitosis. Centrosomes can also act as basal bodies, nucleating the growth of cilia or flagella. Even less understood are the mechanisms used to duplicate membrane‐bound organelles that do not contain DNA. These include organelles involved in the secretory pathway such as the endoplasmic reticulum and the Golgi apparatus. This review will summarize the current knowledge of Golgi biogenesis in simple eukaryotic organisms, in particular, two protozoan parasites, Toxoplasma gondii and Trypanosoma brucei.

A role of autophagy in Trypanosoma brucei cell death

The early branching eukaryote Trypanosoma brucei contains functional autophagy machinery that allows regulated degradation of its own cellular components. In this study, we examined the function of two Atg8 genes, TbAtg8.1 and TbAtg8.2, in starvation‐induced autophagosome formation and cell death in procyclic T. brucei. Upon starvation, both TbAtg8.1 and TbAtg8.2 localize to punctate structures characteristic of autophagosomes as shown by fluorescence and electron microscopy, and wortmannin and chloroquine treatments. While TbAtg8.1 depletion has no detectable effects on TbAtg8.2 recruitment to autophagosomes, TbAtg8.2 depletion greatly reduced the autophagosome relocation of TbAtg8.1. Depletion of TbAtg8.1 and 8.2, individually or together, promote cell survival under starvation conditions. Taken together, these observations confirm the presence of an autophagy‐related cell death pathway in T. brucei, where TbAtg8.1 and TbAtg8.2 play essential but distinct roles in autophagosome formation and cell death.